Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene

Introduction

MtDNA depletion syndromes (MDS) are a genetically and clinically heterogeneous group of autosomal recessive disorders, characterized by low mtDNA levels in specific tissues. These syndromes are due to defects in mtDNA maintenance caused by mutations in nuclear genes involved in nucleotide synthesis (TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, MPV17 and TYMP) or mtDNA replication (POLG and C10orf2). They are classified as myopathic, encephalomyopathic, hepatocerebral or neurogastrointestinal. ¹ The myopathic MDS usually appear in the first year of life and consist of feeding difficulties, failure to thrive, hypotonia and muscle weakness. The disease progresses into a severe myopathy and death because of respiratory failure, but some patients survive into their adulthood with a milder myopathy. ² In all cases, there is a severe reduction in mtDNA content which leads usually to combined respiratory chain complexes deficiency.

Currently, 37 mutations have been reported in the TK2 gene (thymidine kinase 2, mitochondrial; MIM #188250) in 35 patients with the myopathic form of MDS. ³ Mutations in this gene result in loss of thymidine kinase (EC 2.7.1.21) activity in mitochondria, which is responsible for the phosphorylation of pyrimidine nucleosides inside the mitochondria as part of the salvage pathway and as a consequence in a very severe reduction in mtDNA content.

We describe here a patient with myopathy, deficiency of MRC complex III and mtDNA depletion, in whom we detected by whole-exome sequencing (WES) mutation in the well-characterized homozygous mutation T108M in TK2 gene.

Material and Methods

Patient

The 7-year-old patient is the only daughter of non-consanguineous healthy parents. The patient was born at gestational age 38 weeks following uneventful pregnancy and vaginal delivery. She developed normally until the age of 6 months when failure to thrive was observed. Developmental milestones were age-appropriate before 2 years old when she presented an impaired gait with frequent falls. Serum creatine kinase (CK) levels were 236 IU/L at 3 (ref 34–145 IU/L), 389 IU/L at 4, 939 U/L at 5 and 1100 IU/L at 6 years old. Lactacidaemia levels were repeatedly normal. Nerve conduction velocities were normal, and electromyography revealed myogenic abnormalities. At 5 years of age, the physical examination revealed muscle proximal weakness, mild facial weakness and normal deep tendon reflexes. She was able to walk with a waddling gait but was unable to climb upstairs without railings. Gowers’ sign was positive. Distal muscles were preserved and fine motor abilities were normal. Ocular movements were normal and no ptosis was observed. Cerebellar and cognitive functions were preserved. Renal function test showed increased uric acid excretion and hyperaminoaciduria. Magnetic resonance imaging (MRI) of the brain, cardiac evaluation and liver function test were normal. MRI of the patient’s lower pelvis showed involvement of adductor brevis and magnus muscles (Figure 1(a)). The lower limbs at thigh level showed mild-moderate and symmetric fatty replacement of the adductors and muscles of posterior compartment (adductor magnus, biceps femoris, semimembranosus and semitendinosus) (Figure 1(b)), whereas the muscles at lower leg level were spared (Figure 1(c)). Muscle biopsy obtained from the quadriceps showed numerous ragged-red fibers (Figure 2(a)) that were hyper-reactive with succinate dehydrogenase (Figure 2(b)) and were cytochrome-c oxidase (COX)-deficient (Figure 2(c)). COX-deficient fibers accounted for about 10% of all fibers. Frequent necrotic fibers, some with myofagocitosis, and scattered regenerative fibers were also identified. Marked type I fiber predominance was also observed (Figure 2(d)). The activities of MRC complexes in skeletal muscle showed a deficiency of complex III.

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Figure 1.

MRI studies of the patient’s muscle sample. Axial T1 MR images of the patient’s lower limbs at two different levels (lower pelvis, thighs and lower legs). a, The MR images of lower pelvis show involvement of adductor brevis (AB) and magnus (AM) muscles. b, This image shows decreased volume and fatty replacement consistent with mild to moderate involvement of posterior muscles of the thigh: adductor magnus (AM), short and long heads of biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles. There is preservation of sartorius, gracilis, adductor longus, rectus femoris and vasti muscles. c, This MRI T1 transverse images show sparing of muscles in lower legs.

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Figure 2.

Histological and genetic studies. a, Hematoxylin-eosin (HE) staining shows numerous ragged-red fibers (RRF) (*), necrotic fibers, some with myofagocitosis (arrow) and regenerative fibers. b, Succinate dehydrogenase (SDH) staining shows “ragged-blue” fibers that are hyper-reactive. c, Cytochrome-c oxidase (COX) stain shows reduced enzyme activity in several fibers (COX-deficient fibers). d, Staining for myosin adenosine triphosphatase at pH 9.4 (ATPase 9.4) showed mostly fiber-type 1 (fiber-type 1 stained light and type 2 dark). (Objective magnification is indicated in each case. Ocular magnification is x10.) e, Long-range PCR analysis of muscle DNA. MtDNA fragment of 7.3 kb was amplified from total muscle DNA: lane 1, positive control with multiple mtDNA deletions; lane 2, patient with no mtDNA deletions and lane 3, healthy control. M: GeneRuler 1-kb DNA Ladder (life technology).

The DNA analysis in muscle by quantitative polymerase chain reaction (PCR) showed a mtDNA content of 18% (severe mtDNA depletion), whereas the analysis by Southern blot and by long-range PCR did not show mtDNA deletions (Figure 2(e)).

Results

DNA from the patient was subjected to next-generation sequencing of whole-exome to elucidate the molecular basis of the disease. The complete mtDNA sequence was obtained from the WES data described before, ⁴ and no pathogenic variations were detected. In the patient, the SNVs and indels (see “WES variants” file in Supplemental Material) relative to the reference nuclear genome were prioritized. One homozygous variant (NM_004614.4:c.323 C>T, p.T108M) in TK2 gene (thymidine kinase 2, mitochondrial) was obtained after filtering variants that were rare, predicted to have a deleterious impact on protein function and fit to a recessive model of inheritance. ⁵ This variant is described as likely pathogenic by using in silico analysis software (SIFT, PolyPhen2, MutPred and Mutation Taster). Subsequent analysis of blood DNA from the patient’s mother and father confirmed transmission of both mutated alleles.

Discussion

We describe here a patient presenting with a slowly progressing form of myopathic MDS (milder myopathy), and associated with severe mtDNA depletion and absence of mtDNA deletions in skeletal muscle.

In our experience, the use of a targeted NGS panel of 13 nuclear genes (including TK2 gene) involved in the maintenance of the mtDNA integrity for MDS genetic diagnosis has not been as good as we expected (2 patients were diagnosed in a cohort of 23 pediatric patients showing muscle mtDNA levels <30% respect to aged-matched controls). For this reason, the patient was included in a research program of genetic diagnosis by WES. First, the complete mtDNA sequence was obtained from the WES data and no pathogenic variations were detected. So, we could rule out any contribution of somatic mtDNA mutations to the phenotype. Later, the exome analysis of nuclear-encoded genes uncovered a previously reported homozygous pathogenic mutation (T108M) in TK2 gene. ³ This homozygous mutation has been linked to myopathic MDS in 8 patients (3 of them siblings) with very different evolution. The three siblings presented the phenotype in the first year of life and died by the age of 3 with respiratory failure. ⁶ The age of the other 5 patients at the moment of the publication was 44, 31, ² 22, ⁷ 33 and 30 ⁸ years old, respectively. The patient of 44 years old was wheelchair bound and ventilator dependent, and the 31 years old had inability to walk for more than 15 min and moderate respiratory insufficiency. ² The 22-year-old patient was also wheelchair bound with muscle, bulbar, facial and axial weakness. ⁷ The 33-year-old patient had exercise difficulties, muscle weakness, myopathy and pelvic and scapular limb-girdle atrophy, while the 30 years old had dysphagia, muscle weakness and respiratory dysfunction. ⁸ However, the clinical presentation, laboratory data, morphological studies and respiratory chain activities were similar in both groups of patients. The only difference is the percentage of mtDNA, that in the severe cases was <20% and in the milder cases >30%.

Although the onset of myopathic MDS due to the homozygous mutation T108M in TK2 gene takes place in the first years of life, as seen before, the severity appears to correlate with the residual mtDNA content. Thus, the patients with <20% of mtDNA content had a more severe evolution of the disease and died at 3 years old, ⁶ while patients with >30% of mtDNA reached adulthood.^2,7 The patient described here, although have only 18% of mtDNA presents a slower and less severe evolution than those in the former group, being currently 7 years old. Therefore, this report expanded the phenotypic spectrum associated with T108M mutation in TK2 gene to include less severe clinical presentation with very low mtDNA content.