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Termglinchan et al

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Clinical/ScientificNotes

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Thanes Termglinchan,MD
Seito Hisamatsu, MD
Junko Ohmori, PhD
Hiroshi Suzumura, MD,PhD
Noriko Sumitomo, MD
George Imataka, MD,PhD
Osamu Arisaka, MD,PhD
Nobuyuki Murakami,MD, PhD
Narihiro Minami
Ishiyama Akihiko, MD
Masayuki Sasaki, MD,PhD
Yuichi Goto, MD, PhD
Satoru Noguchi, PhD
Ikuya Nonaka, MD, PhD
Satomi Mitsuhashi, MD,PhD
Ichizo Nishino, MD,PhD
Neurol Genet
2016;2:e95; doi: 10.1212/
NXG.0000000000000095

F1

Supplemental data
at Neurology.org/ng

NOVEL TK2 MUTATIONS AS A CAUSE OF
DELAYED MUSCLE MATURATION IN mtDNA
DEPLETION SYNDROMERecessive mutations in TK2 cause a severe mitochondrial DNA depletion syndrome (MDS),1 characterized by severe myopathy from early infancy.
Recent reports have suggested a wider clinical spectrum including encephalomyopathic form.1,2 We
report a patient with infantile-onset fatal encephalomyopathy presenting with extreme muscle fiberimmaturity.

half and markedly disorganized myofibrils in theremainder. Large satellite cells with prominent nuclei
having dense heterochromatin were found in 14% of
the 100 fibers examined (figure 1F). Immunohistochemistry for developmental (MHCd:Leica) and neonatal myosin heavy chain (MHCn:Leica) was
expressed in 24% and 50% of the muscle fibers,
respectively (figures 1D and e-2 and e-3), while inage-matched controls, they made up less than 1% byboth stains (figures 1E and e-2 and 3), reflecting
a marked delay in muscle differentiation.
Whole-exome sequencing revealed 2 novel mutations of TK2 genes: c.125-1G.C substitution in
intron 1 and c.574A.G, p.R192G (GenBank Accession Number: NM_004614.4) (figure 1, G and H).
There were no relevant variants in mitochondrial genesthat can explain the disease. In support of the claim that
this missense mutation causes the disease, the mutation
at the same amino acid position R192K has been reported to cause the disease.3 This amino acid is wellpreserved among species (figure e-4). Sequencing ofcDNA from the patient’s muscle tissue demonstrated
that transcript from the allele carrying the missense
variant was only expressed (figure 1G). The intronicmutation might cause aberrant splicing and nonsensemediated mRNA decay. We concluded that these mutations are compound heterozygous and pathogenic,
although we could not obtain the parents’ DNA.
Mitochondrial respiratory chain enzyme activities
were markedly decreased in skeletal muscle (figuree-5). Quantitative PCR revealed that mitochondrial
DNA (mtDNA) in skeletal muscle was markedly
reduced to 8% (figure e-6).

Patient. The patient was a girl was born to nonconsanguineous healthy Japanese parents at 38 gestationalweeks. Soon after birth, she began to have seizures andrespiratory distress, requiring antiepileptic agents andmechanical ventilation. Physical examination showedfacial weakness with narrow high-arched palate,generalized hypotonia, and muscle weakness. Creatine
kinase level was 7,709 IU/L at 2 days of age. Venous
lactate concentrations were normal. Muscle CT
showed generalized muscle atrophy and fat tissue
replacement. Cardiac echogram revealed hypertrophic
cardiomyopathy. MRI of the brain displayed
lissencephaly, thin corpus callosum and ventricular
dilatation, and cerebellar hypoplasia. Serial brain CT
scans at 3 days (figure 1A) and 1 month (figure 1B)of age revealed rapid brain atrophy with markedly
dilated ventricles. She had progressive generalizedmuscle weakness and atrophy. She died at 10 monthsof age due to congestive heart failure. Permission forautopsy was not obtained.
Biopsy of the biceps brachii at the age of 4 months
revealed that most of the fibers were extremely small,measuring 4–6 mm in diameter. There was marked
endomysial fibrosis without necrotic or regeneratingfibers (figure 1C). Cytochrome c oxidase activity was
diffusely defective (figure e-1 at Neurology.org/ng).
Peripheral nerve bundles in the muscle were well
myelinated. There was no fiber-type grouping andgroup atrophy suggesting peripheral nerve involvement. On electron microscopy, almost all fibers weresmall, measuring less than 5 mm in diameter. None of
the fibers appeared to be normal, showing 2 kinds ofmajor abnormalities: fibers filled with markedly proliferated mitochondria with lipid droplets in about

Discussion. Although TK2 mutations are known tocause myopathy, some patients, including ours, had
CNS abnormalities.1,2,4 A recent article1 reported 2brothers with rapidly progressive brain atrophy similar to our patient, whose muscles showed fat tissue
proliferation. It is not known why the phenotypicvariation occurs in TK2 mutations while mtDNA
depletion levels are similar. Of interest, R192K mutation in the same amino acid was reported previously
in an MDS patient with pure myopathic form,
although the mtDNA was decreased to 8% as inour patient (8%).3 Thus, we suspect that there may
be a phenotype-genotype correlation in this disease.

Neurology.org/ng© 2016 American Academy of Neurologyª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.

1AU2Figure 1Brain imaging, muscle patholgy, immunohistochemistry, electron microscopy, and molecular genetic characterization(A, B) Brain CT at age 3 days (A) and 1 month (B) shows rapidly progressive brain atrophy and ventricular dilatation. (C) In muscle biopsy stained with hematoxylin and eosin stain, most of the fibers are extremely small and embedded in fibrous tissue. (D) On immunohistochemistry for MHCd, developmental MHC
positive fibers stained green are markedly increased in the patient (D.a) but not in controls (D.b). (E) Two representative abnormal fibers on electron microscopy. The left fiber is filled with mitochondria (encircled) and lipid droplets (L). Some mitochondria contain paracrystalline inclusions (inset). The right fibercontains poorly organized myofibrils (encircled). Satellite cell (S). (F, G) Whole-exome sequencing reveals c.574A.G and c.125-1G.C mutations. cDNA ofwhole coding region from muscle shows that only the transcript with missense mutation was expressed. In this transcript, exon 1–2 boundary was normal,suggesting that the other allele possibly with splice site mutation was decayed and not expressed.

The most striking finding in our patient is the delayed muscle maturation in TK2 mutation. Muscle
immaturity was confirmed by numerous positive
2MHCd and MHCn on immunohistochemistry, andpoorly organized myofibrils and increased numbers ofsatellite cells (14%). In normal neonatal muscle,Neurology: Geneticsª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.

satellite cells make up less than 5%. Increased numbers of satellite cells were seen in immature fibers incongenital myopathies and actively regenerating fibers after necrosis. Half of the muscle fibers werefilled with extensively proliferated abnormal mitochondria. Several reports suggest that mitochondrial
biogenesis and activity are significantly increased during myotube differentiation and maturation.4 We
suggest that mitochondrial respiratory chain activities
and mtDNA copy numbers are important factors formuscle maturation.
This is the rare case of TK2-related encephalomyopathic MDS5 with delayed muscle maturation,
thus broadening the spectrum of the disease. It is
worthy to note that MDS with TK2 mutation should
be suspected when a patient shows rapidly progressive
cerebral atrophy and immature skeletal muscle.

comment writing in a genetic testing report for Orphan Net Japan. Dr.
Ishiyama and Dr. Sasaki report no disclosures. Dr. Goto has received
research support from the Japan Agency for Medical Research and Development (AMED) and the National Center of Neurology and Psychiatry(NCNP). Dr. Noguchi holds patent WO 2010/131712A1 and hasreceived research support from Novartis Pharmaceuticals Japan, an
Intramural Research Grant for Neurological and Psychiatric Disordersof NCNP, a grant from the Ministry of Health, Labour, and Welfare,and Health and Labour Sciences Research Grants. Dr. Nonaka andDr. Mitsuhashi report no disclosures. Dr. Nishino has served on thescientific advisory boards of Genzyme, Ultragenyx, and Novartis; hasreceived speaker honoraria from Genzyme; has served on the editorial
boards of Neuromuscular Disorders, Neurology and Clinical
Neuroscience, Therapeutic Advances in Neurological Disorders,the Journal of the Neurological Sciences, Skeletal Muscle, and theJournal of Neuromuscular Diseases; holds a patent for efficaciousagents to pathologic condition due to GNE protein dysfunction; andhas received research support from Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP, JSPS KAKENHI Grant,
the Japan Agency for Medical Research and Development (AMED), andH26-Itaku (Nan)-Ippan-081. Go to Neurology.org/ng for full disclosure
forms. The Article Processing Charge was paid by the authors.

From the Department of Neuromuscular Research (T.T., N. Minami,I.A., S.N., I. Nonaka, S.M., I. Nishino), National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo; Department of Pediatrics (S.H., H.S., G.I., O.A.), Dokkyo MedicalUniversity, Tochigi; Department of Mental Retardation and BirthDefect Research (J.O., Y.G.), Division of Genetic Diagnosis(N. Minami), Department of Laboratory Medicine, National Center
Hospital, NCNP, Tokyo; Department of Child Neurology (N.S.,I.A., M.S.), National Center Hospital, National Center of Neurology
and Psychiatry, Tokyo; Department of Pediatrics (N. Murakami), Dokkyo Medical University Koshigaya Hospital, Saitama; and Departmentof Genome Medicine Development (N. Minami, S.M., I. Nishino),
Medical Genome Center, National Center of Neurology and Psychiatry,Tokyo, Japan.

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Author contributions: Thanes Termglinchan: study concept, analysis
and interpretation of the data and drafting/revising the manuscript.
Seito Hisamatsu, Hiroshi Suzumura, George Imataka, Osamu
Arisaka, and Nobuyuki Murakami: collection of clinical data anddrafting/revising the manuscript. Junko Ohmori, Noriko Sumitomo,Ishiyama Akihiko, Masayuki Sasaki, Satoru Noguchi, Ikuya Nonaka, Satomi Mitsuhashi, and Ichizo Nishino: analysis and interpretation of the data and drafting/revising the manuscript.
Study funding: This study was supported partly by a grantfor Research on Rare and Intractable Diseases (H26-Itaku (Nan)Ippan-081), from Japan Agency for Medical Research and Development (AMED); partly by Intramural Research Grants 26-8, 26-7 for
Neurological and Psychiatric Disorders from the National Center of
Neurology and Psychiatry.
Disclosure: Dr. Termglinchan has received research support from theJapan Agency for Medical Research and Development (AMED), theNational Center of Neurology and Psychiatry, the Epilepsy Society ofThailand, and the Neurological Society of Thailand. Dr. Hisamatsu,
Dr. Ohmori, Dr. Suzumura, Dr. Sumitomo, Dr. Imataka, Dr. Arisaka,and Dr. Murakami report no disclosures. Dr. Minami has completed

Received April 26, 2016. Accepted in final form June 29, 2016.
Correspondence to Dr. Mitsuhashi: smitsuhashi@ncnp.go.jp
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Neurology: Geneticsª 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited.

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