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Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations
Accepted Manuscript Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations S. Roos, U. Lindgren, C. Ehrstedt, A.R. Moslemi, A. Oldfors PII: DOI: Reference:
S0960-8966(14)00135-7 http://dx.doi.org/10.1016/j.nmd.2014.05.009 NMD 2892
To appear in:
Neuromuscular Disorders
Received Date: Revised Date: Accepted Date:
3 February 2014 9 May 2014 20 May 2014
Please cite this article as: Roos, S., Lindgren, U., Ehrstedt, C., Moslemi, A.R., Oldfors, A., Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations, Neuromuscular Disorders (2014), doi: http:// dx.doi.org/10.1016/j.nmd.2014.05.009
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Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations. Roos S1, Lindgren U1, Ehrstedt C2, Moslemi AR1, Oldfors A1 1
Department of Pathology, Institute of Biomedicine, The Sahlgrenska Academy at the
University of Gothenburg, Gothenburg, Sweden, 2Department of Women’s and Children’s Health, Uppsala University Children’s Hospital, Uppsala, Sweden
Corresponding author: Sara Roos University of Gothenburg Institute of Biomedicine Department of Pathology Gula Stråket 8 SE-413 45 Gothenburg Sweden Phone: +46 (0)31-342 2887 Fax: +46 (0)31-342 2886 E-mail: [email protected]
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Abstract The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C>T, p.(T111I) and c.156+5G>C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.
Keywords: mitochondrial DNA depletion syndrome, myopathy, thymidine kinase 2, TK2, mitochondrial myopathy
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Introduction The mitochondrial DNA (mtDNA) depletion syndrome (MDS) [1] is a genetically heterogeneous, autosomal recessive, group of disorders that primarily affects proteins involved in the maintenance of the mitochondrial deoxyribonucleotide (dNTP) pool (mutations in DGUOK, RRM2B, SUCLA2, SUCLG1, TK2, and TYMP), proteins essential for mtDNA replication (mutations in C10orf2 and POLG), or proteins with unknown function (mutations in MPV17) [2-4] and is characterized by reduced copy number of mtDNA in the affected tissues. MDS can be either tissue specific or multisystemic and usually presents in infancy with some exceptions [4].
The mitochondrial dNTP pool is separated from the cytosolic pool by the mitochondrial inner membrane and is maintained either by the import of cytosolic dNTPs or through salvaging the deoxynucleosides within the mitochondria. The mitochondrial thymidine kinase (TK2) is one of the two mitochondrial deoxynucleoside kinases, carrying out the first and rate-limiting phosphorylation step of thymidine, deoxycytidine, and deoxyuridine in the mitochondrial salvage pathway [5]. The myopathic form of MDS has been shown to be associated with mutations in the TK2 gene [6] and to this date there are approximately 30 reported TK2 mutations [2, 6-25]. The disease usually manifests as infantile- or childhood-onset, rapidly progressive, myopathy with increased levels of serum creatine kinase (CK). Most patients die of respiratory failure at 1-3 years of age, but some survived more than 10 years [17]. However, recently published reports show that TK2 mutations can cause early-onset mitochondrial myopathy with slower progression and worsening in adulthood [7, 18] or it can present in adulthood [25, 26].
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In this study, we describe the clinical and molecular genetic findings in a patient with mitochondrial DNA depletion due to two novel heterozygous mutations in the TK2 gene. In an attempt to further characterize mtDNA depletion, we analyzed mtDNA in cytochrome c oxidase (COX)-deficient and COX-positive single muscle fibers.
Materials and Methods Case report Our patient was the first child of non-consanguineous healthy parents of Swedish origin. There was no heredity for neuromuscular disorders. He was born at term after an uneventful pregnancy and delivery. Gross and fine motor skills, as well as psychosocial development was normal during the first 18 months, with the ability to sit and walk independently at 7 and 14 months respectively. After 18 months of age, the parents noted increasing difficulties in walking and climbing stairs, until he finally lost the ability to walk at 22 months of age. At this age he was referred for investigation because of hypotonia and weakness. Besides muscle weakness, which was predominantly proximal, no other clinical findings were noted. Laboratory investigation showed elevated CK-levels (13.0-26.0 µkat/L, reference interval < 5.0 µkat/L) and high P-lactate levels (3.1-4.8 mmol/L, reference interval 0.8-2.0 mmol/L). Neurophysiological studies with neurography and electromyography including repetitive nerve stimulation were performed without any specific findings, supporting the clinical suspicion of a neuromuscular disease. Genetic testing of the SMN1-gene was normal. There were no signs of other organ involvement and kidney, liver and cardiac function were normal. A muscle biopsy was performed and one month after the initial clinical evaluation (at 23 months of age) a diagnosis of mitochondrial myopathy was made. At this point treatment with coenzyme Q10 (5mg/kg/d) and asparaginase (200 mg/kg/d) was started in addition to optimizing the number and quality of calories (120-130% of recommended daily energy
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intake). Clinical follow-up at 25 months of age demonstrated no further progression of muscle weakness. Instead, the patient had regained muscle strength and was able to walk unaided for short distances, although clear proximal muscle weakness, with lordosis and pelvic weakness, was still observed. His cognitive skills have not been formally assessed, but appeared normal.
The clinical improvement seen after initiating treatment with coenzyme Q10, asparaginase and nutritional support (a gastrostomy was provided) was maintained for 7 months until the age of 32 months. After this, a rapid progression of muscle weakness was noted. As a result of profound muscle weakness the patient became wheelchair-bound and respiratory failure was noted during episodes of respiratory tract infections. No other organ involvement was seen and cardiac function remained normal upon investigation with echocardiography. At 42 months of age (3.5 years) he died of respiratory failure.
At autopsy, the lungs showed bronchopneumonia and atelectasis of the inferior lobes. The heart was slightly enlarged. Histological evaluation of the heart showed no evidence of cardiomyopathy or cytochrome c oxidase (COX)-deficiency. The macroscopic and light microscopic findings of the brain were normal, except for a slight periventricular edema. The liver was normal except for signs of acute congestion. M. rectus femoris, m. intercostalis, and the diaphragm showed atrophy with increased interstitial fat and connective tissue. No signs of involvement of other organs were seen. Morphological and molecular genetic analyses At 22 months of age, muscle biopsy was performed and specimens from the vastus lateralis muscle were investigated by morphological and histochemical analyses as previously described [27]. A postmortem was performed when tissue samples from the liver, heart and
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skeletal muscle were collected. Tissue collection and the following analyses were carried out after informed consent. Quantitative real-time PCR (qPCR) Total DNA was isolated from fresh-frozen muscle, and fresh-frozen postmortem tissue samples of muscle, heart, and liver using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) as described in the protocol provided by the manufacturer. Quantitation of mtDNA was determined by TaqMan probe-based quantitative real-time PCR (qPCR) with one probe on the mitochondrial genome and another probe on the nuclear genome, as previously described [28]. Briefly, the nuclear gene CCR5 was selected to serve as the nuclear genome control and the Hs99999149_s1 primer/probe set was used (TaqMan Gene Expression Assays; Life Technologies, Carlsbad, CA, USA). mtDNA primers (forward nt 3782-3806 and reverse nt 3846-3826) and a FAM-labeled probe (nt 3808-3824) were chosen from the ND1-region, a region that is rarely deleted. qPCR reactions were performed in 20 µl mixtures containing 24 ng DNA, 900 nM of each primer, and 200 nM probe. The mtDNA copy number was determined by the mtDNA/nDNA ratio. The patients’ muscle mtDNA levels were then compared to those of seven age-matched controls. Sequence analysis of the TK2 gene and mtDNA Total DNA was extracted from frozen skeletal muscle or from blood. All 10 exons and exonintron boundaries of the TK2 gene were amplified by PCR using intronic primers [20] and sequenced (GenBank accession nr NM_004614.4). Sequencing of the entire mtDNA was performed as described [29]. To rule out large-scale deletions the minor and major arc of mtDNA were amplified by long-range PCR as described [29].
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mRNA expression analysis Total RNA was isolated from frozen muscle using the RNAqueous-4PCR kit (Life Technologies) and first strand cDNA synthesis was performed using Ready-To-Go YouPrime First-Strand Beads from GE Healthcare (Piscataway, NJ, USA). Oligonucleotide primers for TK2 were: forward: 5’-TCC CGA TAA AGA ACA GGA AAA-3’; reverse: 5’CAG GAT TGG TCC GAA GGT AA-3’. Variant analysis To evaluate the incidence of the two novel TK2 variants, a 408-bp fragment covering the c.332C>T variant was amplified by PCR using a forward mismatch primer (5’-TGC CTC TCG CTG GGG TCT TAC GCT GAA GA-3’) and a reverse primer (5’-TTT CCA GGT GAT GCT TCC GCT G-3’). This AC>GA mismatch introduces a unique restriction site for the endonuclease BbsI (New England Biolabs, Ipswich, MA, USA) in the wild-type fragment, and digestion results in two products of 376 and 32 bp. Forward primer 5’-GAA CAG GAA AAA GAG AAA AAA TCA GTG TTA A-3’ and reverse primer 5’-CTT TG ACCT TCA GCC TCA CAC-3’ amplify a 542-bp region covering the c.156+5 G>C variant. This variant contains a unique restriction site for the endonuclease HpaI (New England Biolabs), and digestion results in two products of 513 and 29 bp. In total, 200 chromosomes from healthy blood donors were studied. qPCR of mtDNA in single fibers To further characterize mtDNA depletion, we studied the relative amount of mtDNA depletion in single cells. Single muscle fibers were isolated with a tungsten needle from 16µm thick frozen muscle sections from the vastus lateralis muscle that had been subjected to combined COX and succinate dehydrogenase (SDH) enzyme histochemical staining, as previously described [27]. Single COX-deficient and COX-positive fibers were lysed for 10
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min at 65°C in 5 µl of a freshly made buffer containing 200 mM KOH and 50 mM dithiothreitol (DTT) to which an internal positive control DNA (final concentration 1X, qPCR Internal Positive Control DNA template, Eurogentec, Seraing, Belgium) had been added to be used as an exogenous reference. Then, 5 µl of a neutralizing solution (900 mM Tris-HCl, pH 8.3; 300 mM KCl; 200 mM HCl) was added. All samples were then diluted 1:2 with dH2O to a final volume of 20 µl. The qPCR reaction was run in duplicate, and the level of mtDNA depletion was calculated using the ND1/external DNA ratio. The thermocycling conditions used were; a 20 s hold at 95°; followed by 45 cycles of 95°C for 1 s and 60°C for 20 s on a StepOnePlus cycler (Life Technologies). Fibers of approximately the same size were isolated and in total, 63 single fibers from the patient (33 COX-positive and 30 COX-negative) and 60 single fibers from an age-matched, healthy control were quantified using the assay. Samples with estimated mtDNA content within one SD of the no-template control were rejected. A total of 25 COX-positive and 25 COX-deficient fibers from the patient, and 50 fibers from an age-matched control without evidence of muscle disease were analyzed and included in the results.
Results Morphological and molecular genetic analyses The muscle biopsy demonstrated variability in muscle fiber diameter, with evidence of degenerated fibers, some of which were necrotic (Fig. 1A). Gomori trichrome staining showed mitochondrial proliferation with the presence of numerous ragged-red fibers (Fig. 1B) and small vacuoles were present in many fibers due to lipid accumulation. More than 50% of the fibers were COX-negative (Fig. 1C). Ultrastructural studies showed fibers with a massive accumulation of mitochondria with abnormal cristae. In some fibers the myofibrils were
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completely replaced by abnormal mitochondria, some of them being greatly enlarged (Fig. 1D).
qPCR analysis demonstrated depletion of mtDNA in skeletal muscle tissue, with mtDNA levels at 15% of seven aged-matched controls at age 22 months and 29% of seven agematched controls at age 3.5 years. At 3.5 years, the material from the patient was 96 h post mortem, however, due to lack of material, the age-matched controls were not post mortem. Analysis of postmortem liver tissue demonstrated relative amounts of mtDNA at 25% of one control liver tissue from one adult. The control material was from a liver resection for removal of metastases. No age-matched, postmortem, control liver tissue was available, but it has previously been shown that the mtDNA content in normal liver tissue does not vary with age [30]. The relative mtDNA levels in postmortem heart tissue from the patient were compared with heart muscle tissue from five individuals (13 – 42 years of age) who had undergone cardiac transplantation due to various cardiomyopathies, as no age-matched control tissue was available. The mtDNA content in the patient was 25% of that of these controls. It has however been shown that there is a five-fold increase in mtDNA copy number in the heart from newborns to teen-age [31], suggesting that there was no mtDNA depletion in cardiac tissue of our patient.
Sequencing analysis of the TK2 gene revealed two novel, heterozygous variants: a c.156+5G>C substitution in intron 2 (Fig. 2A) and a c.332C>T transition in exon 5 (Fig. 2B), the latter is predicted to result in a change of a conserved threonine to isoleucine at position 111 p.(T111I) (Fig. 2C). The father carried the c.332C>T mutation and the mother carried the c.156+5G>C mutation (Fig. 2D). Sequencing of cDNA prepared from mRNA of the patient’s muscle tissue demonstrated that only the allele carrying the missense variant was expressed
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(Fig. 2E). The intronic mutation may cause aberrant splicing and nonsense mediated mRNA decay but this was not further analyzed. Neither of the two variants were observed in 200 control chromosomes nor reported in 13,006 alleles in the Exome Variant Server (NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (URL: http://evs.gs.washington.edu/EVS/) [01, 2014]). No point mutations or deletions were found in muscle mtDNA of the patient.
To further characterize the mtDNA depletion disorder, single muscle fibers with normal- and COX-deficient activity were isolated from the patient. Control DNA was added to the lysis buffer before DNA was extracted from the single fibers, and this DNA was then used as an exogenous reference. Single muscle fibers were also isolated in the same manner from a normal, age-matched control, without any evidence of muscle or mitochondrial disease. COXnegative fibers showed a mean relative mtDNA content of 58% compared with COX-positive fibers (Mann-Whitney U test, P < 0.01, n = 25, Fig. 3). The relative amount of mtDNA in the patient was highly reduced in both COX-negative and COX-positive fibers when compared with the age-matched control. When pooling the relative mtDNA values in COX-negative and COX-positive fibers and comparing them with fibers from a control sample, the relative mtDNA levels in fibers of the patient were 14% of the control’s fibers (n = 50). This amount correlates well with the amounts found in muscle homogenate (15% of control). Since it previously has been demonstrated that the 3,3’-diaminobenzadine (DAB) reaction product in the COX histochemical reaction may have an inhibitory effect on qPCR assays [32] we performed additional studies on single fibers from serial sections. The COX status of the individual fibers was determined by COX/SDH staining, but dissected and analyzed for mtDNA content in an adjacent section subjected to SDH staining only. Ten COX-negative fibers were pooled and compared with ten pooled COX-positive fibers. The relative mtDNA amount in the pooled COX-negative fibers was 15% of that in the pooled COX-positive fibers.
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Discussion Mitochondrial depletion syndrome (MDS) may be divided into three clinical categories; hepatocerebral, encephalomyopathic, and myopathic MDS, of which the latter group has been associated with mutations in TK2. Myopathic MDS differs from the other forms as it primarily affects muscle tissue, with the typical manifestation of a severe, rapidly progressing myopathy. We report a patient presenting with hypotonia and muscle weakness at 18 months of age. Elevated CK levels together with lactic acidosis, ragged red- and COX-negative fibers in the muscle biopsy suggested mtDNA depletion syndrome caused by TK2-defiency. Quantitative analysis revealed mtDNA depletion and two novel heterozygous variants in the TK2 gene were identified (c.332C>T/p.[T111I] and c.156+5G>C). cDNA analysis showed that only the allele carrying the missense mutation (c.332C>T/p.[T111I]) was expressed. The c.332C>T mutation changes a conserved (Fig. 2C) threonine to an isoleucine. This amino acid is located in the α4 helix [33], which is in close proximity to the active site [15], suggesting that it could be important for nucleoside recognition. Furthermore, threonine is a polar amino acid with a fairly reactive hydroxyl group and a potential phosphorylation site. Replacement of this polar amino acid with a hydrophobic isoleucine with a non-reactive side chain could be damaging to the function of the TK2 protein [34]. A computer model, Polyphen 2 (http://genetics.bwh.harvard.edu/pph/), which predicts the possible impact of an amino acid substitution on the structure and function of a human protein, suggests that a p.T111I mutation is possibly damaging on protein function. These lines of evidence together with the clinical data support the pathogenicity of the patient’s TK2 substitutions.
The mtDNA amount in skeletal muscle in our patient was 15 and 29% of age-matched controls at age 22 months and 3.5 years, respectively, which is below the cut-off level for MDS diagnosis [35]. However, in MDS caused by mutations in other genes, such as RRM2B,
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the mtDNA depletion in skeletal muscle is much more severe, with levels as low as 1-2% [36]. Such severe depletion caused by RRM2B mutations is usually not associated with overt myopathy. In contrast, many patients with moderate mtDNA depletion caused by TK2 mutations, as in our patient, frequently show necrotic and regenerating muscle fibers and elevated serum CK levels that to some extent mimic muscular dystrophy. The reason why mtDNA depletion caused by TK2 mutations is associated with much more severe myopathy than mtDNA depletion associated with RRM2B mutations remains unclear, but suggests that additional factors may be involved. Although TK2-related MDS usually is associated with a pure myopathy, involvement of other organs has been reported, including the liver with increased hepatic transaminases and mtDNA depletion in liver [23]. mtDNA content in postmortem tissue from the patient’s liver was 25% of control adult liver tissue, but no agematched postmortem liver controls were available. The patient’s liver function was judged as normal and the post-mortem histopathological examination showed only signs of slight congestion but no fibrosis or steatosis. There are some reports of heart involvement in MDS [37, 38], but the underlying genetic defects in these cases are not known. We did not get any evidence of heart involvement in our case.
In a previous report, single muscle fibers from two patients with compound heterozygous variants in TK2; T108M and H212N were investigated [39]. It was demonstrated that there was mitochondrial DNA depletion in both COX-deficient and COX-positive muscle fibers in the patients, and that the mtDNA content was lower in COX-deficient fibers than in COXpositive. However, some muscle fibers retained COX-activity despite a very low copy number of mtDNA and some muscle fibers had very low COX-activity and relatively high concentration of mtDNA. Our results are in concordance with this study; we show that the relative amount of mtDNA was lower in the patient when compared with age-matched
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controls. Furthermore, fibers with severe COX-deficiency had a significantly lower relative amount of mtDNA than fibers with residual COX activity but there was a substantial overlap making it difficult to define a definite threshold level for COX deficiency. In other situations, when a heteroplasmic mitochondrial DNA mutation causes COX deficiency, there is usually a clear threshold level for COX-deficiency in single fibers in cross sections, in spite of variability along each fiber. This is not the only difference between myopathy caused by mtDNA mutations and myopathy caused by mtDNA depletion due to TK2 deficiency. There is a considerable degenerative pathology in TK2 deficiency compared with the situation in most myopathies associated with mtDNA point mutations with similar levels of oxidative phosphorylation deficits. These findings suggest that the COX deficiency is associated with mtDNA depletion and that additional factors may contribute to the pathology. Multiple largescale deletions and single point mutations were excluded by LX-PCR analysis and direct sequencing, but the occurrence of small amounts of clonally expanded point mutations and mtDNA deletions in individual muscle fibers, as can be seen in normal ageing [40], was not studied.
A previous study has shown that DAB may have an inhibitory effect on qPCR assays [32]. In this study, the authors found a significant difference between sections stained for SDH activity alone versus those reacted for both COX and SDH. However, in COX-deficient fibers, this effect was not seen due to the lack of the DAB COX reaction product in these cells. Considering this, we still used the double staining procedure in our experiments because of the risk that the difficulty in isolating the correct fibers using only SDH staining would introduce a larger error in this case where more than 50% of the fibers lacked COX activity. Our results based on analyses of pooled single fibers from sections stained for only SDH without DAB support the concept that mtDNA depletion is the main cause for COX
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deficiency in mtDNA depletion caused by TK2 mutations and also supports the notion that the DAB reaction product may result in underestimation of the mtDNA amount in COX-positive fibers.
To this date, some 30 mutations have been found in the TK2 gene [2]. Similar to our patient, most patients were children who presented with muscle weakness and hypotonia [6, 8-17, 2023] and the mean age of onset was 12.4 months. Typically, muscle weakness rapidly progresses leading to death of respiratory failure. Our patient showed a pronounced improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake. The effectiveness of coenzyme Q10 in the treatment of MDS still needs to be confirmed [41], but it was recently shown that MDS is frequently associated with coenzyme Q10 deficiency [42]. Optimizing the number and quality of calories has been shown to improve mitochondrial health in patients with mitochondrial dysfunction [43], suggesting that the nutritional modulation was of importance for the improvement in muscle strength of our patient.
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Acknowledgements This work was supported by the Swedish Research Council (grant number 07122). The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).
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References [1]
Moraes CT, Shanske S, Tritschler HJ, et al. mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am J Hum Genet 1991;48:492-501.
[2]
Poulton J, Hirano M, Spinazzola A, et al. Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1). Biochim Biophys Acta 2009;1792:1109-12.
[3]
Copeland WC. Defects in mitochondrial DNA replication and human disease. Crit Rev Biochem Mol Biol 2012;47:64-74.
[4]
Suomalainen A, Isohanni P. Mitochondrial DNA depletion syndromes--many genes, common mechanisms. Neuromuscul Disord 2010;20:429-37.
[5]
Saada-Reisch A. Deoxyribonucleoside kinases in mitochondrial DNA depletion. Nucleosides Nucleotides Nucleic Acids 2004;23:1205-15.
[6]
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.
[7]
Behin A, Jardel C, Claeys KG, et al. Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum. Neurology 2012;78:644-8.
[8]
Blakely E, He L, Gardner JL, et al. Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy. Neuromuscul Disord 2008;18:557-60.
[9]
Carrozzo R, Bornstein B, Lucioli S, et al. Mutation analysis in 16 patients with mtDNA depletion. Hum Mutat 2003;21:453-4.
[10]
Collins J, Bove KE, Dimmock D, Morehart P, Wong LJ, Wong B. Progressive myofiber loss with extensive fibro-fatty replacement in a child with mitochondrial
16
DNA depletion syndrome and novel thymidine kinase 2 gene mutations. Neuromuscul Disord 2009;19:784-7. [11]
Galbiati S, Bordoni A, Papadimitriou D, et al. New mutations in TK2 gene associated with mitochondrial DNA depletion. Pediatr Neurol 2006;34:177-85.
[12]
Gotz A, Isohanni P, Pihko H, et al. Thymidine kinase 2 defects can cause multi-tissue mtDNA depletion syndrome. Brain 2008;131:2841-50.
[13]
Lesko N, Naess K, Wibom R, et al. Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion. Neuromuscul Disord 2010;20:198-203.
[14]
Mancuso M, Filosto M, Bonilla E, et al. Mitochondrial myopathy of childhood associated with mitochondrial DNA depletion and a homozygous mutation (T77M) in the TK2 gene. Arch Neurol 2003;60:1007-9.
[15]
Mancuso M, Salviati L, Sacconi S, et al. Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA. Neurology 2002;59:1197-202.
[16]
Marti R, Nascimento A, Colomer J, et al. Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene. Pediatr Res 2010;68:151-4.
[17]
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.
[18]
Paradas C, Gutierrez Rios P, Rivas E, Carbonell P, Hirano M, DiMauro S. TK2 mutation presenting as indolent myopathy. Neurology 2013;80:504-6.
[19]
Stewart JD, Schoeler S, Sitarz KS, et al. POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts. Biochim Biophys Acta 2011;1812:321-5.
17
[20]
Tulinius M, Moslemi AR, Darin N, Holme E, Oldfors A. Novel mutations in the thymidine kinase 2 gene (TK2) associated with fatal mitochondrial myopathy and mitochondrial DNA depletion. Neuromuscul Disord 2005;15:412-5.
[21]
Vila MR, Segovia-Silvestre T, Gamez J, et al. Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 2003;60:1203-5.
[22]
Wang L, Limongelli A, Vila MR, Carrara F, Zeviani M, Eriksson S. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes. Mol Genet Metab 2005;84:75-82.
[23]
Zhang S, Li FY, Bass HN, et al. Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion. Mol Genet Metab 2010;99:53-7.
[24]
Alberio S, Mineri R, Tiranti V, Zeviani M. Depletion of mtDNA: syndromes and genes. Mitochondrion 2007;7:6-12.
[25]
Tyynismaa H, Sun R, Ahola-Erkkila S, et al. Thymidine kinase 2 mutations in autosomal
recessive
progressive
external
ophthalmoplegia
with
multiple
mitochondrial DNA deletions. Hum Mol Genet 2011;21:66-75. [26]
Alston CL, Schaefer AM, Raman P, et al. Late-onset respiratory failure due to TK2 mutations causing multiple mtDNA deletions. Neurology 2013;81:2051-3.
[27]
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.
[28]
Roos S, Macao B, Fuste JM, et al. Subnormal levels of POLgammaA cause inefficient initiation of light-strand DNA synthesis and lead to mitochondrial DNA deletions and
18
autosomal dominant progressive external ophthalmoplegia. Hum Mol Genet 2013;22:2411-22. [29]
Sofou K, Moslemi AR, Kollberg G, et al. Phenotypic and genotypic variability in Alpers syndrome. Eur J Paediatr Neurol 2012;16:379-89.
[30]
Dimmock D, Tang LY, Schmitt ES, Wong LJ. Quantitative evaluation of the mitochondrial DNA depletion syndrome. Clin Chem 2010;56:1119-27.
[31]
Pohjoismaki JL, Goffart S, Taylor RW, et al. Developmental and pathological changes in the human cardiac muscle mitochondrial DNA organization, replication and copy number. PLoS One 2010;5:e10426.
[32]
Murphy JL, Ratnaike TE, Shang E, et al. Cytochrome c oxidase-intermediate fibres: importance in understanding the pathogenesis and treatment of mitochondrial myopathy. Neuromuscul Disord 2012;22:690-8.
[33]
Johansson K, Ramaswamy S, Ljungcrantz C, et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001;8:616-20.
[34]
Betts MJ, Russel RB. Amino acid properties and consequences of substitutions. In: Barnes M R,Gray, I C ed. Bioinformatics for Geneticists: Wiley, 2002: 289-316.
[35]
Poulton J, Holt IJ. 163rd ENMC International Workshop: nucleoid and nucleotide biology in syndromes of mitochondrial DNA depletion myopathy 12-14 December 2008, Naarden, The Netherlands. Neuromuscul Disord 2009;19:439-43.
[36]
Kollberg G, Darin N, Benan K, et al. A novel homozygous RRM2B missense mutation in association with severe mtDNA depletion. Neuromuscul Disord 2009;19:147-50.
[37]
Marin-Garcia J, Ananthakrishnan R, Goldenthal MJ, Filiano JJ, Perez-Atayde A. Cardiac mitochondrial dysfunction and DNA depletion in children with hypertrophic cardiomyopathy. J Inherit Metab Dis 1997;20:674-80.
19
[38]
Santorelli FM, Gagliardi MG, Dionisi-Vici C, et al. Hypertrophic cardiomyopathy and mtDNA depletion. Successful treatment with heart transplantation. Neuromuscul Disord 2002;12:56-9.
[39]
Durham SE, Bonilla E, Samuels DC, DiMauro S, Chinnery PF. Mitochondrial DNA copy number threshold in mtDNA depletion myopathy. Neurology 2005;65:453-5.
[40]
Fayet G, Jansson M, Sternberg D, et al. Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function. Neuromuscul Disord 2002;12:484-93.
[41]
Sacconi S, Trevisson E, Salviati L, et al. Coenzyme Q10 is frequently reduced in muscle of patients with mitochondrial myopathy. Neuromuscul Disord 2010;20:44-8.
[42]
Montero R, Grazina M, Lopez-Gallardo E, et al. Coenzyme Q(1)(0) deficiency in mitochondrial DNA depletion syndromes. Mitochondrion 2013;13:337-41.
[43]
Morava E, Rodenburg R, van Essen HZ, De Vries M, Smeitink J. Dietary intervention and oxidative phosphorylation capacity. J Inherit Metab Dis 2006;29:589.
20
21
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Figure legends Fig. 1. Enzymehistochemical and ultrastructural analyses of muscle. Muscle biopsy from the vastus lateralis muscle showing variation in muscle fiber size as well as mitochondrial proliferation. H&E (A). Many fibers may be classified as ragged-red fibers. Gomori trichrome (B). Cytochrome c oxidase (COX)-succinate dehydrogenase double staining shows profound COX deficiency, with more than 50% of the fibers lacking COX activity (blue fibers) (C). Electron microscopy shows a megamitochondrion with abnormal cristae and an inclusion (D). Many of the muscle fibers of the patient were completely filled with giant abnormal mitochondria as demonstrated in the figure.
Fig. 2. TK2 gene analysis. The patient had two novel heterozygous variations in TK2; a c.156+5G>C transition in intron 2 (A) and a c.332C>T/p.(T111I) substitution in exon 5 (B). The substituted amino acid residue (depicted with a red box) is conserved across vertebrate species (USCS genome browser) (C). Pedigree of family shows the affected individual (solid symbol) (D). Analysis of TK2 cDNA revealed that only the allele with the amino acid substitution is expressed (E).
Fig. 3. mtDNA content in single muscle fibers. Relative amounts of mtDNA in single muscle fibers isolated from 16-µm-thick sections from the patient (COX-negative fibers n = 25 and COX-positive fibers n = 25) and from an agematched control (n = 50) were measured by qPCR. Internal positive control DNA (‘IPC DNA’) had been added to the lysis buffer and served as an exogenous standard. The amount of mtDNA in the patient was reduced in both COX-negative and COX-positive fibers when compared with the age-matched control. The relative amount of mtDNA was reduced more
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than twofold in the COX-negative fibers when compared with COX-positive fibers. Values are median and interquartile range. **P < 0.01, Mann-Whitney U test.
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Abstract The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C>T, p.(T111I) and c.156+5G>C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.
Keywords: mitochondrial DNA depletion syndrome, myopathy, thymidine kinase 2, TK2, mitochondrial myopathy
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S0960-8966(14)00135-7 http://dx.doi.org/10.1016/j.nmd.2014.05.009 NMD 2892
To appear in:
Neuromuscular Disorders
Received Date: Revised Date: Accepted Date:
3 February 2014 9 May 2014 20 May 2014
Please cite this article as: Roos, S., Lindgren, U., Ehrstedt, C., Moslemi, A.R., Oldfors, A., Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations, Neuromuscular Disorders (2014), doi: http:// dx.doi.org/10.1016/j.nmd.2014.05.009
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Mitochondrial DNA depletion in single fibers in a patient with novel TK2 mutations. Roos S1, Lindgren U1, Ehrstedt C2, Moslemi AR1, Oldfors A1 1
Department of Pathology, Institute of Biomedicine, The Sahlgrenska Academy at the
University of Gothenburg, Gothenburg, Sweden, 2Department of Women’s and Children’s Health, Uppsala University Children’s Hospital, Uppsala, Sweden
Corresponding author: Sara Roos University of Gothenburg Institute of Biomedicine Department of Pathology Gula Stråket 8 SE-413 45 Gothenburg Sweden Phone: +46 (0)31-342 2887 Fax: +46 (0)31-342 2886 E-mail: [email protected]
1
Abstract The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C>T, p.(T111I) and c.156+5G>C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.
Keywords: mitochondrial DNA depletion syndrome, myopathy, thymidine kinase 2, TK2, mitochondrial myopathy
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Introduction The mitochondrial DNA (mtDNA) depletion syndrome (MDS) [1] is a genetically heterogeneous, autosomal recessive, group of disorders that primarily affects proteins involved in the maintenance of the mitochondrial deoxyribonucleotide (dNTP) pool (mutations in DGUOK, RRM2B, SUCLA2, SUCLG1, TK2, and TYMP), proteins essential for mtDNA replication (mutations in C10orf2 and POLG), or proteins with unknown function (mutations in MPV17) [2-4] and is characterized by reduced copy number of mtDNA in the affected tissues. MDS can be either tissue specific or multisystemic and usually presents in infancy with some exceptions [4].
The mitochondrial dNTP pool is separated from the cytosolic pool by the mitochondrial inner membrane and is maintained either by the import of cytosolic dNTPs or through salvaging the deoxynucleosides within the mitochondria. The mitochondrial thymidine kinase (TK2) is one of the two mitochondrial deoxynucleoside kinases, carrying out the first and rate-limiting phosphorylation step of thymidine, deoxycytidine, and deoxyuridine in the mitochondrial salvage pathway [5]. The myopathic form of MDS has been shown to be associated with mutations in the TK2 gene [6] and to this date there are approximately 30 reported TK2 mutations [2, 6-25]. The disease usually manifests as infantile- or childhood-onset, rapidly progressive, myopathy with increased levels of serum creatine kinase (CK). Most patients die of respiratory failure at 1-3 years of age, but some survived more than 10 years [17]. However, recently published reports show that TK2 mutations can cause early-onset mitochondrial myopathy with slower progression and worsening in adulthood [7, 18] or it can present in adulthood [25, 26].
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In this study, we describe the clinical and molecular genetic findings in a patient with mitochondrial DNA depletion due to two novel heterozygous mutations in the TK2 gene. In an attempt to further characterize mtDNA depletion, we analyzed mtDNA in cytochrome c oxidase (COX)-deficient and COX-positive single muscle fibers.
Materials and Methods Case report Our patient was the first child of non-consanguineous healthy parents of Swedish origin. There was no heredity for neuromuscular disorders. He was born at term after an uneventful pregnancy and delivery. Gross and fine motor skills, as well as psychosocial development was normal during the first 18 months, with the ability to sit and walk independently at 7 and 14 months respectively. After 18 months of age, the parents noted increasing difficulties in walking and climbing stairs, until he finally lost the ability to walk at 22 months of age. At this age he was referred for investigation because of hypotonia and weakness. Besides muscle weakness, which was predominantly proximal, no other clinical findings were noted. Laboratory investigation showed elevated CK-levels (13.0-26.0 µkat/L, reference interval < 5.0 µkat/L) and high P-lactate levels (3.1-4.8 mmol/L, reference interval 0.8-2.0 mmol/L). Neurophysiological studies with neurography and electromyography including repetitive nerve stimulation were performed without any specific findings, supporting the clinical suspicion of a neuromuscular disease. Genetic testing of the SMN1-gene was normal. There were no signs of other organ involvement and kidney, liver and cardiac function were normal. A muscle biopsy was performed and one month after the initial clinical evaluation (at 23 months of age) a diagnosis of mitochondrial myopathy was made. At this point treatment with coenzyme Q10 (5mg/kg/d) and asparaginase (200 mg/kg/d) was started in addition to optimizing the number and quality of calories (120-130% of recommended daily energy
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intake). Clinical follow-up at 25 months of age demonstrated no further progression of muscle weakness. Instead, the patient had regained muscle strength and was able to walk unaided for short distances, although clear proximal muscle weakness, with lordosis and pelvic weakness, was still observed. His cognitive skills have not been formally assessed, but appeared normal.
The clinical improvement seen after initiating treatment with coenzyme Q10, asparaginase and nutritional support (a gastrostomy was provided) was maintained for 7 months until the age of 32 months. After this, a rapid progression of muscle weakness was noted. As a result of profound muscle weakness the patient became wheelchair-bound and respiratory failure was noted during episodes of respiratory tract infections. No other organ involvement was seen and cardiac function remained normal upon investigation with echocardiography. At 42 months of age (3.5 years) he died of respiratory failure.
At autopsy, the lungs showed bronchopneumonia and atelectasis of the inferior lobes. The heart was slightly enlarged. Histological evaluation of the heart showed no evidence of cardiomyopathy or cytochrome c oxidase (COX)-deficiency. The macroscopic and light microscopic findings of the brain were normal, except for a slight periventricular edema. The liver was normal except for signs of acute congestion. M. rectus femoris, m. intercostalis, and the diaphragm showed atrophy with increased interstitial fat and connective tissue. No signs of involvement of other organs were seen. Morphological and molecular genetic analyses At 22 months of age, muscle biopsy was performed and specimens from the vastus lateralis muscle were investigated by morphological and histochemical analyses as previously described [27]. A postmortem was performed when tissue samples from the liver, heart and
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skeletal muscle were collected. Tissue collection and the following analyses were carried out after informed consent. Quantitative real-time PCR (qPCR) Total DNA was isolated from fresh-frozen muscle, and fresh-frozen postmortem tissue samples of muscle, heart, and liver using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) as described in the protocol provided by the manufacturer. Quantitation of mtDNA was determined by TaqMan probe-based quantitative real-time PCR (qPCR) with one probe on the mitochondrial genome and another probe on the nuclear genome, as previously described [28]. Briefly, the nuclear gene CCR5 was selected to serve as the nuclear genome control and the Hs99999149_s1 primer/probe set was used (TaqMan Gene Expression Assays; Life Technologies, Carlsbad, CA, USA). mtDNA primers (forward nt 3782-3806 and reverse nt 3846-3826) and a FAM-labeled probe (nt 3808-3824) were chosen from the ND1-region, a region that is rarely deleted. qPCR reactions were performed in 20 µl mixtures containing 24 ng DNA, 900 nM of each primer, and 200 nM probe. The mtDNA copy number was determined by the mtDNA/nDNA ratio. The patients’ muscle mtDNA levels were then compared to those of seven age-matched controls. Sequence analysis of the TK2 gene and mtDNA Total DNA was extracted from frozen skeletal muscle or from blood. All 10 exons and exonintron boundaries of the TK2 gene were amplified by PCR using intronic primers [20] and sequenced (GenBank accession nr NM_004614.4). Sequencing of the entire mtDNA was performed as described [29]. To rule out large-scale deletions the minor and major arc of mtDNA were amplified by long-range PCR as described [29].
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mRNA expression analysis Total RNA was isolated from frozen muscle using the RNAqueous-4PCR kit (Life Technologies) and first strand cDNA synthesis was performed using Ready-To-Go YouPrime First-Strand Beads from GE Healthcare (Piscataway, NJ, USA). Oligonucleotide primers for TK2 were: forward: 5’-TCC CGA TAA AGA ACA GGA AAA-3’; reverse: 5’CAG GAT TGG TCC GAA GGT AA-3’. Variant analysis To evaluate the incidence of the two novel TK2 variants, a 408-bp fragment covering the c.332C>T variant was amplified by PCR using a forward mismatch primer (5’-TGC CTC TCG CTG GGG TCT TAC GCT GAA GA-3’) and a reverse primer (5’-TTT CCA GGT GAT GCT TCC GCT G-3’). This AC>GA mismatch introduces a unique restriction site for the endonuclease BbsI (New England Biolabs, Ipswich, MA, USA) in the wild-type fragment, and digestion results in two products of 376 and 32 bp. Forward primer 5’-GAA CAG GAA AAA GAG AAA AAA TCA GTG TTA A-3’ and reverse primer 5’-CTT TG ACCT TCA GCC TCA CAC-3’ amplify a 542-bp region covering the c.156+5 G>C variant. This variant contains a unique restriction site for the endonuclease HpaI (New England Biolabs), and digestion results in two products of 513 and 29 bp. In total, 200 chromosomes from healthy blood donors were studied. qPCR of mtDNA in single fibers To further characterize mtDNA depletion, we studied the relative amount of mtDNA depletion in single cells. Single muscle fibers were isolated with a tungsten needle from 16µm thick frozen muscle sections from the vastus lateralis muscle that had been subjected to combined COX and succinate dehydrogenase (SDH) enzyme histochemical staining, as previously described [27]. Single COX-deficient and COX-positive fibers were lysed for 10
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min at 65°C in 5 µl of a freshly made buffer containing 200 mM KOH and 50 mM dithiothreitol (DTT) to which an internal positive control DNA (final concentration 1X, qPCR Internal Positive Control DNA template, Eurogentec, Seraing, Belgium) had been added to be used as an exogenous reference. Then, 5 µl of a neutralizing solution (900 mM Tris-HCl, pH 8.3; 300 mM KCl; 200 mM HCl) was added. All samples were then diluted 1:2 with dH2O to a final volume of 20 µl. The qPCR reaction was run in duplicate, and the level of mtDNA depletion was calculated using the ND1/external DNA ratio. The thermocycling conditions used were; a 20 s hold at 95°; followed by 45 cycles of 95°C for 1 s and 60°C for 20 s on a StepOnePlus cycler (Life Technologies). Fibers of approximately the same size were isolated and in total, 63 single fibers from the patient (33 COX-positive and 30 COX-negative) and 60 single fibers from an age-matched, healthy control were quantified using the assay. Samples with estimated mtDNA content within one SD of the no-template control were rejected. A total of 25 COX-positive and 25 COX-deficient fibers from the patient, and 50 fibers from an age-matched control without evidence of muscle disease were analyzed and included in the results.
Results Morphological and molecular genetic analyses The muscle biopsy demonstrated variability in muscle fiber diameter, with evidence of degenerated fibers, some of which were necrotic (Fig. 1A). Gomori trichrome staining showed mitochondrial proliferation with the presence of numerous ragged-red fibers (Fig. 1B) and small vacuoles were present in many fibers due to lipid accumulation. More than 50% of the fibers were COX-negative (Fig. 1C). Ultrastructural studies showed fibers with a massive accumulation of mitochondria with abnormal cristae. In some fibers the myofibrils were
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completely replaced by abnormal mitochondria, some of them being greatly enlarged (Fig. 1D).
qPCR analysis demonstrated depletion of mtDNA in skeletal muscle tissue, with mtDNA levels at 15% of seven aged-matched controls at age 22 months and 29% of seven agematched controls at age 3.5 years. At 3.5 years, the material from the patient was 96 h post mortem, however, due to lack of material, the age-matched controls were not post mortem. Analysis of postmortem liver tissue demonstrated relative amounts of mtDNA at 25% of one control liver tissue from one adult. The control material was from a liver resection for removal of metastases. No age-matched, postmortem, control liver tissue was available, but it has previously been shown that the mtDNA content in normal liver tissue does not vary with age [30]. The relative mtDNA levels in postmortem heart tissue from the patient were compared with heart muscle tissue from five individuals (13 – 42 years of age) who had undergone cardiac transplantation due to various cardiomyopathies, as no age-matched control tissue was available. The mtDNA content in the patient was 25% of that of these controls. It has however been shown that there is a five-fold increase in mtDNA copy number in the heart from newborns to teen-age [31], suggesting that there was no mtDNA depletion in cardiac tissue of our patient.
Sequencing analysis of the TK2 gene revealed two novel, heterozygous variants: a c.156+5G>C substitution in intron 2 (Fig. 2A) and a c.332C>T transition in exon 5 (Fig. 2B), the latter is predicted to result in a change of a conserved threonine to isoleucine at position 111 p.(T111I) (Fig. 2C). The father carried the c.332C>T mutation and the mother carried the c.156+5G>C mutation (Fig. 2D). Sequencing of cDNA prepared from mRNA of the patient’s muscle tissue demonstrated that only the allele carrying the missense variant was expressed
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(Fig. 2E). The intronic mutation may cause aberrant splicing and nonsense mediated mRNA decay but this was not further analyzed. Neither of the two variants were observed in 200 control chromosomes nor reported in 13,006 alleles in the Exome Variant Server (NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (URL: http://evs.gs.washington.edu/EVS/) [01, 2014]). No point mutations or deletions were found in muscle mtDNA of the patient.
To further characterize the mtDNA depletion disorder, single muscle fibers with normal- and COX-deficient activity were isolated from the patient. Control DNA was added to the lysis buffer before DNA was extracted from the single fibers, and this DNA was then used as an exogenous reference. Single muscle fibers were also isolated in the same manner from a normal, age-matched control, without any evidence of muscle or mitochondrial disease. COXnegative fibers showed a mean relative mtDNA content of 58% compared with COX-positive fibers (Mann-Whitney U test, P < 0.01, n = 25, Fig. 3). The relative amount of mtDNA in the patient was highly reduced in both COX-negative and COX-positive fibers when compared with the age-matched control. When pooling the relative mtDNA values in COX-negative and COX-positive fibers and comparing them with fibers from a control sample, the relative mtDNA levels in fibers of the patient were 14% of the control’s fibers (n = 50). This amount correlates well with the amounts found in muscle homogenate (15% of control). Since it previously has been demonstrated that the 3,3’-diaminobenzadine (DAB) reaction product in the COX histochemical reaction may have an inhibitory effect on qPCR assays [32] we performed additional studies on single fibers from serial sections. The COX status of the individual fibers was determined by COX/SDH staining, but dissected and analyzed for mtDNA content in an adjacent section subjected to SDH staining only. Ten COX-negative fibers were pooled and compared with ten pooled COX-positive fibers. The relative mtDNA amount in the pooled COX-negative fibers was 15% of that in the pooled COX-positive fibers.
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Discussion Mitochondrial depletion syndrome (MDS) may be divided into three clinical categories; hepatocerebral, encephalomyopathic, and myopathic MDS, of which the latter group has been associated with mutations in TK2. Myopathic MDS differs from the other forms as it primarily affects muscle tissue, with the typical manifestation of a severe, rapidly progressing myopathy. We report a patient presenting with hypotonia and muscle weakness at 18 months of age. Elevated CK levels together with lactic acidosis, ragged red- and COX-negative fibers in the muscle biopsy suggested mtDNA depletion syndrome caused by TK2-defiency. Quantitative analysis revealed mtDNA depletion and two novel heterozygous variants in the TK2 gene were identified (c.332C>T/p.[T111I] and c.156+5G>C). cDNA analysis showed that only the allele carrying the missense mutation (c.332C>T/p.[T111I]) was expressed. The c.332C>T mutation changes a conserved (Fig. 2C) threonine to an isoleucine. This amino acid is located in the α4 helix [33], which is in close proximity to the active site [15], suggesting that it could be important for nucleoside recognition. Furthermore, threonine is a polar amino acid with a fairly reactive hydroxyl group and a potential phosphorylation site. Replacement of this polar amino acid with a hydrophobic isoleucine with a non-reactive side chain could be damaging to the function of the TK2 protein [34]. A computer model, Polyphen 2 (http://genetics.bwh.harvard.edu/pph/), which predicts the possible impact of an amino acid substitution on the structure and function of a human protein, suggests that a p.T111I mutation is possibly damaging on protein function. These lines of evidence together with the clinical data support the pathogenicity of the patient’s TK2 substitutions.
The mtDNA amount in skeletal muscle in our patient was 15 and 29% of age-matched controls at age 22 months and 3.5 years, respectively, which is below the cut-off level for MDS diagnosis [35]. However, in MDS caused by mutations in other genes, such as RRM2B,
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the mtDNA depletion in skeletal muscle is much more severe, with levels as low as 1-2% [36]. Such severe depletion caused by RRM2B mutations is usually not associated with overt myopathy. In contrast, many patients with moderate mtDNA depletion caused by TK2 mutations, as in our patient, frequently show necrotic and regenerating muscle fibers and elevated serum CK levels that to some extent mimic muscular dystrophy. The reason why mtDNA depletion caused by TK2 mutations is associated with much more severe myopathy than mtDNA depletion associated with RRM2B mutations remains unclear, but suggests that additional factors may be involved. Although TK2-related MDS usually is associated with a pure myopathy, involvement of other organs has been reported, including the liver with increased hepatic transaminases and mtDNA depletion in liver [23]. mtDNA content in postmortem tissue from the patient’s liver was 25% of control adult liver tissue, but no agematched postmortem liver controls were available. The patient’s liver function was judged as normal and the post-mortem histopathological examination showed only signs of slight congestion but no fibrosis or steatosis. There are some reports of heart involvement in MDS [37, 38], but the underlying genetic defects in these cases are not known. We did not get any evidence of heart involvement in our case.
In a previous report, single muscle fibers from two patients with compound heterozygous variants in TK2; T108M and H212N were investigated [39]. It was demonstrated that there was mitochondrial DNA depletion in both COX-deficient and COX-positive muscle fibers in the patients, and that the mtDNA content was lower in COX-deficient fibers than in COXpositive. However, some muscle fibers retained COX-activity despite a very low copy number of mtDNA and some muscle fibers had very low COX-activity and relatively high concentration of mtDNA. Our results are in concordance with this study; we show that the relative amount of mtDNA was lower in the patient when compared with age-matched
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controls. Furthermore, fibers with severe COX-deficiency had a significantly lower relative amount of mtDNA than fibers with residual COX activity but there was a substantial overlap making it difficult to define a definite threshold level for COX deficiency. In other situations, when a heteroplasmic mitochondrial DNA mutation causes COX deficiency, there is usually a clear threshold level for COX-deficiency in single fibers in cross sections, in spite of variability along each fiber. This is not the only difference between myopathy caused by mtDNA mutations and myopathy caused by mtDNA depletion due to TK2 deficiency. There is a considerable degenerative pathology in TK2 deficiency compared with the situation in most myopathies associated with mtDNA point mutations with similar levels of oxidative phosphorylation deficits. These findings suggest that the COX deficiency is associated with mtDNA depletion and that additional factors may contribute to the pathology. Multiple largescale deletions and single point mutations were excluded by LX-PCR analysis and direct sequencing, but the occurrence of small amounts of clonally expanded point mutations and mtDNA deletions in individual muscle fibers, as can be seen in normal ageing [40], was not studied.
A previous study has shown that DAB may have an inhibitory effect on qPCR assays [32]. In this study, the authors found a significant difference between sections stained for SDH activity alone versus those reacted for both COX and SDH. However, in COX-deficient fibers, this effect was not seen due to the lack of the DAB COX reaction product in these cells. Considering this, we still used the double staining procedure in our experiments because of the risk that the difficulty in isolating the correct fibers using only SDH staining would introduce a larger error in this case where more than 50% of the fibers lacked COX activity. Our results based on analyses of pooled single fibers from sections stained for only SDH without DAB support the concept that mtDNA depletion is the main cause for COX
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deficiency in mtDNA depletion caused by TK2 mutations and also supports the notion that the DAB reaction product may result in underestimation of the mtDNA amount in COX-positive fibers.
To this date, some 30 mutations have been found in the TK2 gene [2]. Similar to our patient, most patients were children who presented with muscle weakness and hypotonia [6, 8-17, 2023] and the mean age of onset was 12.4 months. Typically, muscle weakness rapidly progresses leading to death of respiratory failure. Our patient showed a pronounced improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake. The effectiveness of coenzyme Q10 in the treatment of MDS still needs to be confirmed [41], but it was recently shown that MDS is frequently associated with coenzyme Q10 deficiency [42]. Optimizing the number and quality of calories has been shown to improve mitochondrial health in patients with mitochondrial dysfunction [43], suggesting that the nutritional modulation was of importance for the improvement in muscle strength of our patient.
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Acknowledgements This work was supported by the Swedish Research Council (grant number 07122). The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the WHI Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010).
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References [1]
Moraes CT, Shanske S, Tritschler HJ, et al. mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am J Hum Genet 1991;48:492-501.
[2]
Poulton J, Hirano M, Spinazzola A, et al. Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase, POLG1). Biochim Biophys Acta 2009;1792:1109-12.
[3]
Copeland WC. Defects in mitochondrial DNA replication and human disease. Crit Rev Biochem Mol Biol 2012;47:64-74.
[4]
Suomalainen A, Isohanni P. Mitochondrial DNA depletion syndromes--many genes, common mechanisms. Neuromuscul Disord 2010;20:429-37.
[5]
Saada-Reisch A. Deoxyribonucleoside kinases in mitochondrial DNA depletion. Nucleosides Nucleotides Nucleic Acids 2004;23:1205-15.
[6]
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.
[7]
Behin A, Jardel C, Claeys KG, et al. Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum. Neurology 2012;78:644-8.
[8]
Blakely E, He L, Gardner JL, et al. Novel mutations in the TK2 gene associated with fatal mitochondrial DNA depletion myopathy. Neuromuscul Disord 2008;18:557-60.
[9]
Carrozzo R, Bornstein B, Lucioli S, et al. Mutation analysis in 16 patients with mtDNA depletion. Hum Mutat 2003;21:453-4.
[10]
Collins J, Bove KE, Dimmock D, Morehart P, Wong LJ, Wong B. Progressive myofiber loss with extensive fibro-fatty replacement in a child with mitochondrial
16
DNA depletion syndrome and novel thymidine kinase 2 gene mutations. Neuromuscul Disord 2009;19:784-7. [11]
Galbiati S, Bordoni A, Papadimitriou D, et al. New mutations in TK2 gene associated with mitochondrial DNA depletion. Pediatr Neurol 2006;34:177-85.
[12]
Gotz A, Isohanni P, Pihko H, et al. Thymidine kinase 2 defects can cause multi-tissue mtDNA depletion syndrome. Brain 2008;131:2841-50.
[13]
Lesko N, Naess K, Wibom R, et al. Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion. Neuromuscul Disord 2010;20:198-203.
[14]
Mancuso M, Filosto M, Bonilla E, et al. Mitochondrial myopathy of childhood associated with mitochondrial DNA depletion and a homozygous mutation (T77M) in the TK2 gene. Arch Neurol 2003;60:1007-9.
[15]
Mancuso M, Salviati L, Sacconi S, et al. Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA. Neurology 2002;59:1197-202.
[16]
Marti R, Nascimento A, Colomer J, et al. Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene. Pediatr Res 2010;68:151-4.
[17]
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.
[18]
Paradas C, Gutierrez Rios P, Rivas E, Carbonell P, Hirano M, DiMauro S. TK2 mutation presenting as indolent myopathy. Neurology 2013;80:504-6.
[19]
Stewart JD, Schoeler S, Sitarz KS, et al. POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts. Biochim Biophys Acta 2011;1812:321-5.
17
[20]
Tulinius M, Moslemi AR, Darin N, Holme E, Oldfors A. Novel mutations in the thymidine kinase 2 gene (TK2) associated with fatal mitochondrial myopathy and mitochondrial DNA depletion. Neuromuscul Disord 2005;15:412-5.
[21]
Vila MR, Segovia-Silvestre T, Gamez J, et al. Reversion of mtDNA depletion in a patient with TK2 deficiency. Neurology 2003;60:1203-5.
[22]
Wang L, Limongelli A, Vila MR, Carrara F, Zeviani M, Eriksson S. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes. Mol Genet Metab 2005;84:75-82.
[23]
Zhang S, Li FY, Bass HN, et al. Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion. Mol Genet Metab 2010;99:53-7.
[24]
Alberio S, Mineri R, Tiranti V, Zeviani M. Depletion of mtDNA: syndromes and genes. Mitochondrion 2007;7:6-12.
[25]
Tyynismaa H, Sun R, Ahola-Erkkila S, et al. Thymidine kinase 2 mutations in autosomal
recessive
progressive
external
ophthalmoplegia
with
multiple
mitochondrial DNA deletions. Hum Mol Genet 2011;21:66-75. [26]
Alston CL, Schaefer AM, Raman P, et al. Late-onset respiratory failure due to TK2 mutations causing multiple mtDNA deletions. Neurology 2013;81:2051-3.
[27]
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.
[28]
Roos S, Macao B, Fuste JM, et al. Subnormal levels of POLgammaA cause inefficient initiation of light-strand DNA synthesis and lead to mitochondrial DNA deletions and
18
autosomal dominant progressive external ophthalmoplegia. Hum Mol Genet 2013;22:2411-22. [29]
Sofou K, Moslemi AR, Kollberg G, et al. Phenotypic and genotypic variability in Alpers syndrome. Eur J Paediatr Neurol 2012;16:379-89.
[30]
Dimmock D, Tang LY, Schmitt ES, Wong LJ. Quantitative evaluation of the mitochondrial DNA depletion syndrome. Clin Chem 2010;56:1119-27.
[31]
Pohjoismaki JL, Goffart S, Taylor RW, et al. Developmental and pathological changes in the human cardiac muscle mitochondrial DNA organization, replication and copy number. PLoS One 2010;5:e10426.
[32]
Murphy JL, Ratnaike TE, Shang E, et al. Cytochrome c oxidase-intermediate fibres: importance in understanding the pathogenesis and treatment of mitochondrial myopathy. Neuromuscul Disord 2012;22:690-8.
[33]
Johansson K, Ramaswamy S, Ljungcrantz C, et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nat Struct Biol 2001;8:616-20.
[34]
Betts MJ, Russel RB. Amino acid properties and consequences of substitutions. In: Barnes M R,Gray, I C ed. Bioinformatics for Geneticists: Wiley, 2002: 289-316.
[35]
Poulton J, Holt IJ. 163rd ENMC International Workshop: nucleoid and nucleotide biology in syndromes of mitochondrial DNA depletion myopathy 12-14 December 2008, Naarden, The Netherlands. Neuromuscul Disord 2009;19:439-43.
[36]
Kollberg G, Darin N, Benan K, et al. A novel homozygous RRM2B missense mutation in association with severe mtDNA depletion. Neuromuscul Disord 2009;19:147-50.
[37]
Marin-Garcia J, Ananthakrishnan R, Goldenthal MJ, Filiano JJ, Perez-Atayde A. Cardiac mitochondrial dysfunction and DNA depletion in children with hypertrophic cardiomyopathy. J Inherit Metab Dis 1997;20:674-80.
19
[38]
Santorelli FM, Gagliardi MG, Dionisi-Vici C, et al. Hypertrophic cardiomyopathy and mtDNA depletion. Successful treatment with heart transplantation. Neuromuscul Disord 2002;12:56-9.
[39]
Durham SE, Bonilla E, Samuels DC, DiMauro S, Chinnery PF. Mitochondrial DNA copy number threshold in mtDNA depletion myopathy. Neurology 2005;65:453-5.
[40]
Fayet G, Jansson M, Sternberg D, et al. Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function. Neuromuscul Disord 2002;12:484-93.
[41]
Sacconi S, Trevisson E, Salviati L, et al. Coenzyme Q10 is frequently reduced in muscle of patients with mitochondrial myopathy. Neuromuscul Disord 2010;20:44-8.
[42]
Montero R, Grazina M, Lopez-Gallardo E, et al. Coenzyme Q(1)(0) deficiency in mitochondrial DNA depletion syndromes. Mitochondrion 2013;13:337-41.
[43]
Morava E, Rodenburg R, van Essen HZ, De Vries M, Smeitink J. Dietary intervention and oxidative phosphorylation capacity. J Inherit Metab Dis 2006;29:589.
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Figure legends Fig. 1. Enzymehistochemical and ultrastructural analyses of muscle. Muscle biopsy from the vastus lateralis muscle showing variation in muscle fiber size as well as mitochondrial proliferation. H&E (A). Many fibers may be classified as ragged-red fibers. Gomori trichrome (B). Cytochrome c oxidase (COX)-succinate dehydrogenase double staining shows profound COX deficiency, with more than 50% of the fibers lacking COX activity (blue fibers) (C). Electron microscopy shows a megamitochondrion with abnormal cristae and an inclusion (D). Many of the muscle fibers of the patient were completely filled with giant abnormal mitochondria as demonstrated in the figure.
Fig. 2. TK2 gene analysis. The patient had two novel heterozygous variations in TK2; a c.156+5G>C transition in intron 2 (A) and a c.332C>T/p.(T111I) substitution in exon 5 (B). The substituted amino acid residue (depicted with a red box) is conserved across vertebrate species (USCS genome browser) (C). Pedigree of family shows the affected individual (solid symbol) (D). Analysis of TK2 cDNA revealed that only the allele with the amino acid substitution is expressed (E).
Fig. 3. mtDNA content in single muscle fibers. Relative amounts of mtDNA in single muscle fibers isolated from 16-µm-thick sections from the patient (COX-negative fibers n = 25 and COX-positive fibers n = 25) and from an agematched control (n = 50) were measured by qPCR. Internal positive control DNA (‘IPC DNA’) had been added to the lysis buffer and served as an exogenous standard. The amount of mtDNA in the patient was reduced in both COX-negative and COX-positive fibers when compared with the age-matched control. The relative amount of mtDNA was reduced more
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than twofold in the COX-negative fibers when compared with COX-positive fibers. Values are median and interquartile range. **P < 0.01, Mann-Whitney U test.
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Abstract The mitochondrial DNA (mtDNA) depletion syndrome is a genetically heterogeneous group of diseases caused by nuclear gene mutations and secondary reduction in mtDNA copy number. We describe a patient with progressive muscle weakness and increased creatine kinase and lactate levels. Muscle weakness was first noted at age 1.5 years and he died of respiratory failure and bronchopneumonia at age 3.5 years. The muscle biopsy showed dystrophic features with ragged red fibers and numerous cytochrome c oxidase (COX)negative fibers. qPCR analysis demonstrated depletion of mtDNA and sequence analysis of the mitochondrial thymidine kinase 2 (TK2) gene revealed two novel heterozygous variants, c.332C>T, p.(T111I) and c.156+5G>C. Quantitative analysis of mtDNA in single muscle fibers demonstrated that COX-deficient fibers showed more pronounced depletion of mtDNA when compared with fibers with residual COX activity (P < 0.01, n = 25). There was no evidence of manifestations from other organs than skeletal muscle although there was an apparent reduction of mtDNA copy number also in liver. The patient showed a pronounced, albeit transient, improvement in muscle strength after onset of treatment with coenzyme Q10, asparaginase, and increased energy intake, suggesting that nutritional modulation may be a therapeutic option in myopathic mtDNA depletion syndrome.
Keywords: mitochondrial DNA depletion syndrome, myopathy, thymidine kinase 2, TK2, mitochondrial myopathy
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