Clinica Chimica Acta 448 (2015) 211–214

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Clinica Chimica Acta
journal homepage: www.elsevier.com/locate/clinchim

Novel POLG mutation in a patient with sensory ataxia, neuropathy,
ophthalmoparesis and stroke
Ching-Wan Lam a,⁎, Chun-Yiu Law a, Wai-Kwan Siu b, Cheuk-Wing Fung c, Man-Mut Yau d, Kwai-Fun Huen d,
Hencher Han-Chih Lee b, Chloe Miu Mak b
a

Department of Pathology, The University of Hong Kong, Hong Kong, China
Department of Pathology, Princess Margaret Hospital, Hong Kong, China
Department of Pediatrics and Adolescent Medicine, Queen Mary Hospital, Hong Kong, China
d
Department of Pediatrics, Tseung Kwan O Hospital, Hong Kong, China
b
c

a r t i c l e

i n f o

Article history:
Received 8 May 2015
Received in revised form 22 June 2015
Accepted 22 June 2015
Available online 11 July 2015
Keywords:
POLG
Stroke
Sensory ataxia
Neuropathy
Ophthalmoparesis

a b s t r a c t
Background: Clinical diagnosis of POLG-related disorders can be challenging because the phenotypic spectrums
are heterogeneous which can mimic different types of mitochondrial disorders.
Case: We report a case of POLG-related disorder in an 18 y Chinese girl who had been diagnosed as MELAS
syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) for the past 8 y.
She first presented at 10 y with sudden onset of headache, repeated focal seizures and visual loss, complicated
with residual sensory and motor neuropathy, ophthalmoparesis and cortical blindness. MRI brain showed
extensive cytotoxic edema and ischemia in bilateral parietal–occipital lobes. Mutation analysis for common
point mutations in the mitochondrial DNA and muscle biopsy was negative. She was referred to us for mitochondrial whole genome analysis. However, no pathogenic variants can be determined. We initiated further genetic
analysis for POLG which confirmed compound heterozygous mutations NM_002693.2:c.925CNT (p.Arg309Cys)
and a novel mutation c.2244GNT (p.Trp748Cys). Both were determined to be pathogenic using in silico analysis.
Conclusions: The novel mutation contributes to the expanding spectrum of disease-causing mutations. A
definitive diagnosis can benefit our patient and also the relatives by avoiding sodium valproate induced liver
toxicity in POLG patients and also the heterozygotes.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Human mitochondrial DNA (mtDNA) is a double-stranded circular
DNA located in the mitochondria inherited in a uniparental maternal
manner. The mtDNA genome is composed of 16,569 base pairs,
encrypting for 37 genes: 22 transfer RNA (tRNA), 13 core proteins in
mitochondrial respiratory complexes and 2 mitochondrial ribosomecoding RNA (rRNA) [1]. Mutations of mtDNA would result in various
clinical disorders, for example, Alpers-Huttenlocher syndrome, ataxia
neuropathy syndromes, chronic progressive external ophthalmoplegia
(CPEO), Kearns–Sayre syndrome, Leber hereditary optic neuropathy
(LHON), Leigh syndrome, mitochondrial myopathy, encephalopathy,
lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy
myopathy sensory ataxia (MEMSA), myoclonic epilepsy with raggedred fibers (MERRF), neurogenic weakness with ataxia and retinitis
pigmentosa (NARP) and Pearson syndrome [2].

⁎ Corresponding author.
E-mail address: ching-wanlam@pathology.hku.hk (C.-W. Lam).

http://dx.doi.org/10.1016/j.cca.2015.06.028
0009-8981/© 2015 Elsevier B.V. All rights reserved.

Both DNA polymerase gamma (encoded by the POLG) [3] and
Twinkle (encoded by the C10orf2) [4] are essential for mtDNA
replication. DNA polymerase gamma is a nuclear-encoded protein
which has both polymerase activity and a proofreading exonuclease
activity for mtDNA [5]. Therefore, patients with POLG mutation can
have similar phenotypes as those patients with inherited mitochondrial
disorders [6]. The first reported case of POLG mutation was described in
2001 in which the patient presented with progressive external
ophthalmoplegia (PEO) [7]. Since then, more POLG-related disorders
had been reported, for example male infertility [8], MERRF [9], a
syndrome of ophthalmoplegia, sensorimotor polyneuropathy, ataxia,
and deafness [10], Alpers' syndrome [11], ataxia without myopathy
[12], premature ovarian failure [13], familial parkinsonism [14], axonal
and demyelinating sensory-motor neuropathy [15], multiple oxidative
phosphorylation deficiencies [16], sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO) [17] and MELAS [18]. The
phenotypic spectrums of POLG-related disorders are heterogeneous.
Therefore, clinical diagnosis of POLG-related disorders is challenging.
Very often, the diagnosis is relied on molecular genetic test for POLG
gene.

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2. Case report
The proband was an 18-y Chinese girl born to non-consanguineous
parents with unremarkable birth history. She enjoyed good past health.
She first presented at 10 y with sudden onset of headache, repeated
focal seizures and loss of vision. She was treated as having acute meningoencephalitis. Seizures were controlled with phenytoin and carbamazepine. Ophthalmological assessment confirmed no structural lesion and
the sudden loss of vision was compatible with cerebral visual impairment.
Her sensorium gradually improved. However, there was no improvement
on her vision. Magnetic resonance imaging (MRI) of the brain showed extensive cytotoxic edema and ischemia in bilateral parietal–occipital lobes
(Fig. 1). The overall MRI features were compatible with acute ischaemia.
However, subsequent magnetic resonance angiogram (MRA) showed
no vascular abnormalities. Electroencephalography (EEG) and microbiology investigations were negative. The patient also had unremarkable
blood acid–base profile. Blood lactate was 1.0 mmol/l (reference interval
(RI): 0.7–2.1). Laboratory testing for plasma amino acid and organic acid
profile and very long chain fatty acid levels was unremarkable. Investigation of cerebral spinal fluid (CSF) protein was normal, CSF glucose was 4.7
mmol/l (paired plasma glucose was 8.5 mmol/l), CSF lactate was 1.5
mmol/l (RI: 0.45–2.1), and CSF pyruvate was 0.07 mmol/l (RI: b0.10).
There was no CSF oligoclonal band. Screening for autoimmune markers
was negative. Muscle biopsy was performed which showed no fiber
necrosis or regeneration, no hyaline change, and no peri-fascicular/
endomysial inflammatory infiltrate and fibrosis. There were no ragged
red fibers. The muscle fibers showed normal succinic dehydrogenase
staining. There was no moth-eaten fiber or sarcoplasmic inclusions.
Electron-microscopy of the muscle tissue showed normal mitochondria
appearance. MELAS syndrome was suspected for this patient. However,
common pathogenic mutations in mtDNA, including m.3243ANG,
m.8344ANG and m.8893TNG were negative. She was given arginine
supplement, vitamin cocktail and anticonvulsants (initially phenytoin
followed by carbamazepine monotherapy).
The patient showed good recovery on sensorium with residual
sensory and motor ataxia. Nerve conduction study performed at the
first admission confirmed sensory and motor neuropathy. However,
her vision was severely impaired with only light perception remained.
Visual evoked potential (VEP) test confirmed conduction delay along
the optic nerve secondary to ischemic optic neuropathy. She also had

limited abduction of both eyes. Subsequently, she had two episodes of
recurrent seizure attacks and hemiparesis about 1 year and 2 years
after the first admission. Serial MRI of the brain showed no new
changes. The patient was followed up regularly with serial blood lactate
monitoring. The blood lactates were all normal except one episode of
mild lactate elevation (2.3 mmol/l). All along, her liver function test
and liver ultrasonography were normal.
3. Materials and methods
3.1. Mitochondrial whole genome sequencing analysis
Blood samples and/or urine sediment samples were collected from
the proband and her unaffected brother and parents after informed
consent. Extraction of DNA from urine sediments was performed
according to the manufacturer procedures using Qiagen DNA extraction
kit (Qiagen). The entire mitochondrial genome was amplified with
genomic DNA extracted from the urine sediment sample of the proband
in 5 overlapping fragments, m.782 to m.8484, m.4502 to m.11527,
m.5197 to m.11524, m.7819 to m.15274 and m.11714 to m.3527 using
long range polymerase chain reaction (PCR). Primer sequences are
available upon request. The long range PCR products were purified
using Agencourt AMPure XP (Beckman Coulter) and quantitated by
Quant-iT Picogreen (Invitrogen). A library was built using the GS Junior
Titanium Rapid Library Prep Kit (Roche Diagnostics) by shearing of the
DNA using nebulization (kit part of the library prep kit) followed by
ligation of Rapid Library Adaptor to the DNA according to the
manufacturer's protocols (454 Life Sciences). The library was amplified
using emulsion PCR with the GS Junior Titanium emPCR Kit (Roche) following the manufacturer's protocol. An input of 2 molecules of library
DNA per capture bead was used. After emulsion PCR, the beads capturing the DNA library were enriched and the enriched beads were
annealed with sequencing primers. The amplified fragments were sequenced using 500,000 enriched beads and 200 cycles with full processing for shotgun or paired end pipeline in the GS Junior Benchtop System
(Roche). Post-sequencing raw data was filtered to eliminate low-quality
sequence data. The reads were mapped to mitochondrial genome using
GS Reference Mapper. Mutational analysis and pipeline used to rule out
variants of unknown significance were based on published American
College of Medical Genetics (ACMG) clinical laboratory standards for
next-generation sequencing [19].
3.2. Mutational analysis using Sanger sequencing for POLG
Methods for PCR and sequencing are described previously [20].
Primer sequences for POLG are available upon request. Sequencing
results of all coding exons and the flanking intronic regions of the
POLG genes were compared with National Center for Biotechnology Information (NCBI) reference sequences NM_002693.2 and NP_002684.1.
The pathogenicity of novel mutation was determined using in silico
analysis, Polymorphism Phenotyping v2 (PolyPhen-2) online, http://
genetics.bwh.harvard.edu/pph2/.
3.3. NMR-based urinalysis
NMR-based urinalysis was acquired using a 600 MHz Bruker NMR
spectrometer using a “noesygppr1d” pulse sequence. Details of the
sample preparation, proton NMR acquisition parameters and positive
identifications of metabolites had been described previously [21,22].
4. Results
4.1. Mutational analysis

Fig. 1. Extensive T2-hyperintense signal over bilateral parietal–occipital areas.

In the mitochondrial whole genome sequencing analysis, 114,779
DNA sequence reads were obtained after processing with quality filters

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213

for c.2244GNT (p.Trp748Cys) and the mother (I-2) was heterozygous
for c.925CNT (p.Arg309Cys). The two disease-causing mutations were
not found in the unaffected brother (II-2) (Fig. 3).
4.2. 1H-NMR-based urinalysis
The 1H NMR-based urinalysis showed increased urine ascorbic acid
and carnitine. The findings were compatible with the drug record of
the proband which was put on L-carnitine and vitamin C supplements.
Overall, the urine metabolic pattern was unremarkable.
5. Discussion
Fig. 2. Pedigree of a POLG-affected family.

with average read length of 457 base-pairs. The percentage of reads
mapped to the mitochondrial genome was 98.69% and the coverage of
the mitochondrial genome was 100%. The average read depth was
400 ×. The mitochondrial whole genome sequencing analysis in the
urine sediment sample of the proband did not detect any point mutations or large mitochondrial deletion.
The pedigree of the family is shown in Fig. 2. Mutation analysis for
POLG gene of the proband (II-1) showed a compound heterozygous mutation NM_002693.2:c.925CNT (p.Arg309Cys) and NM_002693.2:
c.2244GNT (p.Trp748Cys). Homozygous p.Arg309Cys had been previously reported in patient with peripheral neuropathy, progressive external ophthalmoplegia, ataxia and myopathy [23]. The p.Trp748Cys is
a novel mutation. Using PolyPhen-2 prediction, both mutations were
predicted to be probably damaging. The father (I-1) was heterozygous

The phenotype of POLG-related disorders is heterogeneous which
can mimic different types of mitochondria disorders [6,24]. The phenotypes of POLG-related disorders include (i) mitochondrial DNA
depletion syndrome 4A (Alpers type), (ii) mitochondrial DNA depletion
syndrome 4B (MNGIE type); (iii) Mitochondrial recessive ataxia
syndrome (includes SANDO and SCAE); (iv) progressive external
ophthalmoplegia, autosomal dominant and (v) progressive external
ophthalmoplegia, autosomal recessive [25]. Most of the reported POLG
mutations were recessive mutations with few of them being dominant
mutations [26].
The patient in this case report presented with stroke-like episode,
sensory ataxia, neuropathy and ophthalmoplegia, and the mode of
inheritance was autosomal recessive, therefore the overall clinical
features is compatible with SANDO syndrome (sensory ataxic neuropathy, dysarthria and ophthalmoparesis; OMIM #607459). However, she
had no remarkable dysarthria. Indeed, dysarthria can be absent in
patients with SANDO caused by POLG mutations [27]. In a recent study

Fig. 3. Electropherograms showing the POLG mutations in the family.

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of SANDO syndrome, dysarthria was found to be absent in some
patients who carried compound heterozygous pathogenic POLG mutations. Therefore, etiology of dysarthria in SANDO remained inconclusive
[28]. Intriguingly, our patient also presented with stroke-like episode
which was further evident by extensive cytotoxic edema and ischemia
in bilateral parietal–occipital lobes by MRI brain study. The patient
also showed hemiparesis in her subsequent admissions. Stroke-like
episode is not a usual feature in SANDO. This case implies the heterogeneous clinical features of POLG-related disorders. To our knowledge,
none of the known POLG-related syndrome can fit all the clinical
features of our patient.
A definitive diagnosis can benefit our patient and also the relatives by
avoiding sodium valproate induced liver toxicity in POLG patients and also
the heterozygotes [29,30]. Clinician should be aware of POLG-related
disorder in patients with unexplained MELAS-like presentations, neurological deficit, liver dysfunction and suspected mitochondrial diseases.
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