Abstract

Background

   Mutations in the gene encoding thymidine kinase 2 (TK2) result in the
   myopathic form of mitochondrial DNA depletion syndrome which is a
   mitochondrial encephalomyopathy presenting in children. In order to
   unveil some of the mechanisms involved in this pathology and to
   identify potential biomarkers and therapeutic targets we have
   investigated the gene expression profile of human skeletal muscle
   deficient for TK2 using cDNA microarrays.

Results

   We have analysed the whole transcriptome of skeletal muscle from
   patients with TK2 mutations and compared it to normal muscle and to
   muscle from patients with other mitochondrial myopathies. We have
   identified a set of over 700 genes which are differentially expressed
   in TK2 deficient muscle. Bioinformatics analysis reveals important
   changes in muscle metabolism, in particular, in glucose and glycogen
   utilisation, and activation of the starvation response which affects
   aminoacid and lipid metabolism. We have identified those
   transcriptional regulators which are likely to be responsible for the
   observed changes in gene expression.

Conclusion

   Our data point towards the tumor suppressor p53 as the regulator at the
   centre of a network of genes which are responsible for a coordinated
   response to TK2 mutations which involves inflammation, activation of
   muscle cell death by apoptosis and induction of growth and
   differentiation factor 15 (GDF-15) in muscle and serum. We propose that
   GDF-15 may represent a potential novel biomarker for mitochondrial
   dysfunction although further studies are required.

   Keywords: Gene expression, Microarrays, Bioinformatics, Mitochondrial
   DNA, Mitochondrial DNA depletion, Mitochondrial encephalomyopathy,
   Thymidine kinase 2, Skeletal muscle, p53, Apoptosis, GDF-15

Background

   Mitochondrial myopathy and mitochondrial encephalomyopathy are terms
   that define a group of neurological disorders characterized by
   defective oxidative phosphorylation [[72]1-[73]3]. Multiple organs may
   be affected including skeletal muscle, liver and central nervous
   system. The genetics and pathogenesis of mitochondrial disorders are
   complex because of the interaction of the mtDNA and nuclear genomes.
   The mtDNA contains 37 genes encoding 13 mitochondrial proteins. The
   remaining of the over 1500 predicted mitochondrial proteins are encoded
   by the nuclear genome [[74]4]. The majority of these nuclear genes are
   unknown (over 50 are known as disease causative genesmaking it very
   difficult to diagnose at the molecular level mitochondrial disorders
   not due to mtDNA mutations [[75]5,[76]6].

   Mitochondrial DNA depletion syndrome (MDS) is a clinically and
   genetically heterogeneous subgroup of mitochondrial diseases of nuclear
   origin which is characterised by severe reduction of mtDNA content in
   specific tissues, mainly muscle, liver and brain To date 9 nuclear
   genes have been identified as causing MDS which encode proteins
   directly involved in the maintenance of the mitochondrial
   deoxyribonucleoside triphosphate pools (dNTPs) such as Thymidine kinase
   2 (TK2), deoxyguanosine kinase (DGOUK), p53 dependent ribonucleotide
   reductase subunit 2 (RRM2B) and thymidine phosphorylase (TYMP), or in
   mtDNA replication such as mtDNA polymerase γ (POLG) and Twinkle
   (C10orf2). The role of the other three MDS causative gene products
   (MPV17), and the α and β subunits of succinate-CoA ligase (SUCLG1 and
   SUCLA2 respectively) in mtDNA maintenance is less clear [[77]7].

   Clinically, MDS are divided into myopathic, hepatocerebellar or
   encephalomyopathic, depending on which tissues are mainly affected at
   the onset of the symptoms. The myopathic form is most frequently
   associated with mutations in the TK2, SUCLA2 and RRM2B genes. Thymidine
   kinase 2 is responsible for the phosphorylation of pyrimidine
   nucleosides (deoxythymidine, deoxycytosine and deoxyuridine) inside the
   mitochondria as part of the salvage pathway. In contrast, thymidine
   kinase 1 (TK1) participates in the de novo deoxythymidine monophosphate
   (dTMP) synthesis pathway in the cytoplasm which is then imported into
   mitochondria. In most reported cases mutations in TK2 result in almost
   complete absence of enzyme activity and as a consequence in a very
   severe reduction in mtDNA content (>90%) which leads to combined
   respiratory chain complexes deficiency. Myopathic MDS due to TK2
   defects is characterised by neonatal or early onset of hypotonia which
   progresses rapidly into a severe myopathy and infantile death because
   of respiratory failure although there are reports of patients which
   survive longer [[78]8,[79]9]. Electromyography shows myopathic signs;
   creatine kinase levels (CK) are markedly elevated and lactate is
   moderately high. In addition to muscle symptoms patients may also have
   CNS involvement [[80]7,[81]10-[82]12]. At the pathological level,
   muscle biopsies frequently show dystrophic changes with loss of muscle
   fibres, proliferation of connective and adipose tissues, ragged red
   fibers with mitochondria proliferation and profound cytochrome c
   oxidase deficiency and increased intracellular lipid content [[83]10].
   To date, more than 20 missense mutations, deletions and insertions have
   been reported in the TK2 gene. Some of them have been reported in more
   than one non-related families [[84]11,[85]12].

   Two mouse models of TK2 defects have been generated. Tk2 knocked-out
   mice [[86]13,[87]14] suffer growth retardation and hypothermia and die
   within 2–4 weeks of life. They show progressive depletion of mtDNA in
   skeletal muscle, heart and liver. The morphology of skeletal muscle
   seems unaffected whereas cardiac muscle is disorganised and contains
   abnormally structured mitochondria. The authors conclude that Tk2 is
   necessary for deoxythymidine triphosphate (dTTP) synthesis in
   non-replicating cells, whereas fast-replicating cells rely more heavily
   on the de novo synthesis pathway. Thus, as tissues mature, mtDNA
   replication is increasingly affected and mtDNA depletion worsens.
   Detailed analysis of the neurological features of these mice revealed
   an ataxic phenotype with progressive loss of mtDNA content, respiratory
   chain activity and protein content of mitochondrially encoded
   respiratory chain subunits in brain tissue, which is accompanied by
   selective loss of neuronal types [[88]13]. The Tk2 knock-in mice bear
   the human H126N missense mutation and also showed growth retardation
   and had a generalized weakness, tremor and impaired gait [[89]15]. Tk2
   activity was severely reduced in all tissues tested (3% and 1.7% of
   normal in skeletal muscle and brain respectively) confirming that the
   mutation severely disrupts Tk2 function. Brain and spinal cord showed
   the most prominent mtDNA depletion followed by skeletal muscle, heart
   and kidney whereas in contrast to the KO model, liver mtDNA content was
   normal. Activities of the respiratory chain enzymes were selectively
   reduced in brain and spinal cord whereas they were normal in skeletal
   muscle. Histological analysis of skeletal muscle was unremarkable. In
   contrast, the equivalent homozygous mutation in humans resulted in
   severe myopathy [[90]16]. Thus, neither of the two mouse models
   replicates the severe muscle phenotype and pathology seen in humans.

   How the muscle cell responds to mtDNA depletion, impaired oxidative
   phosphorylation and severe loss of energy production and how these
   mechanisms differ between MDS and other mitochondrial myopathies is not
   well understood. A patient was described which survived into adulthood.
   Analysis of his two consecutive muscle biopsies revealed an increase in
   mtDNA content with time which paradoxically was accompanied by a
   decrease in complex IV activity and severe muscle mass and fibre loss
   [[91]8,[92]17]. These findings suggest that there may have been a
   selective loss of the most affected fibres and that a compensatory
   mechanism may be acting in these patients to maintain mtDNA levels in
   surviving muscle fibres. Identification of such compensatory pathways
   would be of great importance to devise potential therapies.

   In order to address some of these questions we undertook global gene
   expression analysis of muscle biopsies from MDS patients with mutations
   in TK2 and compared it with that of patients with mtDNA large deletions
   and aged matched normal muscle. We found a large number of genes whose
   expression was significantly changed which represent the molecular
   signature of TK2 deficient human muscle. Our data show that the
   activation of p53 signalling pathway is relevant to the pathogenesis of
   TK2 defects and show that energy production via glucose and glycogen
   metabolism are significantly impaired at the transcriptional level. In
   contrast, genes known to be part of the starvation response mechanism
   were increased. We describe the over-expression of growth and
   differentiation factor 15 (GDF-15) in muscle and serum and its
   potential use as a novel biomarker of mitochondrial disease.

Results

Patients and microarray

   In the cDNA microarray we included 4 unrelated patients with MDS and
   mutations in TK2 (patients 1, 2, 3 and 4, MDS group), 4 patients with
   large mtDNA deletions as disease control group (patients 5, 6, 7 and 8,
   MDEL group) and 4 aged-matched healthy individuals (normal control
   group) (Table [93]1). Whole genome was analysed using Agilent Human
   8 × 60K arrays. Standard array quality controls were performed showing
   satisfactory results. Unsupervised bi-clustering analysis considering
   the Agilent oligonucleotides with the largest variations across the
   whole experiment showed an appropriate segregation of the different
   samples. Data has been deposited at the National Centre for
   Biotechnology Information Gene Expression Omnibus (GEO) database as GEO
   Series accession number [94]GSE43698.

Table 1.

   Clinical summary of patients (* indicates those patients included in
   the microarray analysis)
   Patient Onset Main clinical symptoms Age at biopsy Genetics Respiratory
   chain enzymes activities Muscle pathology References