Mitochondrial DNA depletion in progressive external ophthalmoplegia caused by POLG1 mutations.
ABSTRACT To investigate two patients with late onset, progressive external ophthalmoplegia (PEO) and sensory peripheral neuropathy.
The patients aged 86 and 50 years were investigated clinically including magnetic resonance imaging of the brain, electrophysiological studies and, in one, skeletal muscle biopsy. Molecular studies included sequencing of the whole coding region of the POLG1 gene and mitochondrial DNA (mtDNA) analysis for deletions and depletion.
Both patients were compound heterozygous for gene encoding the catalytic subunit of the DNA-polymerase gamma (POLG1) mutations. One had the p.737R and p.W748S mutations while the other carried the p.T251I, p.P587L and p.W748S mutations. While these mutations have been previously described, these combinations are novel. mtDNA studies in skeletal muscle showed evidence of multiple deletions and approximately 64% depletion of the mitochondrial genome.
Our findings broaden the genotypic spectrum of POLG-associated PEO and show that in addition to multiple deletions, mtDNA depletion occurs and may contribute to the pathogenesis of this disorder.
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Mitochondrial DNA depletion in progressive
external ophthalmoplegia caused by POLG1
Progressive external ophthalmoplegia (PEO) is a
genetically heterogeneous mitochondrial disorder
characterized by ptosis, external ophthalmoplegia
and a variable degree of proximal muscle weak-
ness. Additional features may include: ataxia,
peripheral neuropathy, deafness, cataract, hypo-
gonadism and Parkinsonism (1–3). Sporadic and
maternally inherited PEO can be caused by single,
heteroplasmic deletions or point mutations of
mitochondrial DNA (mtDNA). Autosomal domi-
nant PEO is caused by mutations in nuclear genes
including POLG1 and POLG2, encoding the
catalytic and accessory subunits of the mtDNA
polymerase gamma, respectively, PEO1 encoding
the mitochondrial helicase twinkle and ANT1 that
encodes the muscle and heart isoforms of the
adenine nucleotide translocator 1. Autosomal
recessive PEO is associated with mutations in
Over 120 pathogenic mutations have been
described in POLG1 and at least 60 of these are
chondrial spinocerebellar ataxia with epilepsy
(MSCAE) (5, 6), sensory ataxia neuropathy dysar-
thria and ophthalmoparesis (SANDO) (1) and
Parkinsonian syndromes (3). POLG1 mutations
also cause Alpers? syndrome (7). At the molecular
level, POLG-related disease is associated with sec-
deletions and quantitative depletion. Tissue-specific
mtDNA depletion affectingthe liver, skeletal muscle
and brain is typically found in patients with Alpers?
syndrome (7, 8). Multiple deletions are mostly
associated with PEO and depletion has not been
reported in these patients (1, 9–13).
Acta Neurol Scand 2009: 120 (Suppl. 189): 38–41
? 2009 John Wiley & Sons A ⁄S
Tzoulis C, Papingji M, Fiskestrand T, Røste LS, Bindoff LA.
Mitochondrial DNA depletion in progressive external ophthalmoplegia
caused by POLG1 mutations.
Acta Neurol Scand 2009: 120 (Suppl. 189): 38–41.
? 2009 John Wiley & Sons A ⁄S.
Objectives – To investigate two patients with late onset, progressive
external ophthalmoplegia (PEO) and sensory peripheral neuropathy.
Materials & Methods – The patients aged 86 and 50 years were
investigated clinically including magnetic resonance imaging of the
brain, electrophysiological studies and, in one, skeletal muscle biopsy.
Molecular studies included sequencing of the whole coding region of
the POLG1 gene and mitochondrial DNA (mtDNA) analysis for
deletions and depletion. Results – Both patients were compound
heterozygous for gene encoding the catalytic subunit of the DNA-
polymerase gamma (POLG1) mutations. One had the p.737R and
p.W748S mutations while the other carried the p.T251I, p.P587L and
p.W748S mutations. While these mutations have been previously
described, these combinations are novel. mtDNA studies in skeletal
muscle showed evidence of multiple deletions and approximately 64%
depletion of the mitochondrial genome. Conclusion – Our findings
broaden the genotypic spectrum of POLG-associated PEO and show
that in addition to multiple deletions, mtDNA depletion occurs and
may contribute to the pathogenesis of this disorder.
C. Tzoulis1,2, M. Papingji1,
T. Fiskestrand3, L. S. Røste4,
L. A. Bindoff1,2
1Department of Neurology, Haukeland University
Hospital, Bergen, Norway;2Department of Clinical
Medicine, University of Bergen, Bergen, Norway;
3Center for Medical Genetics and Molecular Medicine,
Haukeland University Hospital, Bergen, Norway;
4Department of Neurology, Division of Clinical
Neuroscience, Rikshospitalet University Hospital, Oslo,
Key words: progressive external ophthalmoplegia;
POLG; mtDNA; depletion
Laurence Bindoff, Department of Neurology, Haukeland
University Hospital, 5021 Bergen, Norway
Tel.: +47 559 75096
Fax: +47 559 75165
Conflicts of interest: The authors declare no conflicts of
Materials and methods
Patients were evaluated clinically by at least one of
the authors. A skeletal muscle biopsy was per-
formed on patient A1, but patient B1 refused
biopsy. Genomic DNA was extracted from blood
and muscle using standard protocols. POLG1
exons 2–23 were amplified and sequenced using
intronic primers and BigDye Terminator cycle
sequencing kit (v3.1; Applied Biosystems Inc.,
Foster City, CA, USA). mtDNA deletions were
detected by long-range PCR (LPCR) using primers
to generate amplicons of two different lengths: one
of approximately 10 kb (6730–370) and one of
approximately15 kb (1138–16526; primers and
protocol available on request). Amplified products
were electrophoresed through a 0.7% agarose gel,
at 40 V for 240 m. Quantification of the deleted
and total mtDNA content in muscle from patient
A1 and controls was performed by real-time PCR
using TaqMan fluorogenic probes and an Applied
Biosystems 7500 fast sequence detection system.
The percentage of deleted mtDNA was calculated
by comparing amplification within the commonly
deleted ND4 region and rarely deleted ND1 region.
Quantification of total mtDNA was performed by
comparing amplification of ND1 with a nuclear
multi-copy gene (18SrRNA). The primers and
fluorogenic probes used have been described pre-
viously (14). Each reaction was performed in
triplicate and in two independent runs with the
following profile: one cycle at 95?C for 20 s and
then 40 cycles at 95?C for 3 s and 60?C for 30 s.
Threshold cycle numbers (Ct) were calculated with
7500 Fast System SDS software v1.4 (Applied
Biosystems) and the results from the two runs were
averaged. The ND4 ⁄ND1 and ND1 ⁄18S ratios
were calculated from cycle threshold values as
described previously (14).
Patient A1 is a 50-year-old female, born to non-
consanguineous parents who presented with a
5-year history of diplopia, ptosis and muscle fatigue
upon physical activity. Her sister had been operated
for bilateral ptosis, but her parents and grandpar-
ents were reportedly healthy. Clinically she had
bilateral, asymmetrical ptosis and a mild external
ophthalmoplegia affecting horizontal, but not
vertical gaze. Convergence was impaired. The
sensory defect in stocking distribution was seen.
able. Skeletal muscle biopsy showed about 5%
COX-negative fibres, but no ragged-red fibres.
Magnetic resonance imaging (MRI) of the brain
showed mild diffuse leucoencephalopathy with high
apparent diffusion coefficient (ADC; Fig. 1).
Patient B1 is an 86-year-old female with a
previous history of primary hypothyroidism and
bilateral hearing loss of uncertain duration. She
had been operated for bilateral ptosis at 75 years of
age and presented to us with 2–3 years of worsen-
ing diplopia, gait unsteadiness and paresthaesiae in
the distal lower limbs. She was the product of non-
consanguineous parents and reported no similar
symptoms in her family. Clinical examination
revealed asymmetrical ptosis and nearly complete
external ophthalmoplegia with loss of convergence,
oculocephalic reflex and Bell?s reflex. She had
symmetrical distal sensory loss in the lower limbs
and absence of Achilles reflexes, but no sensory
ataxia. Electromyography and nerve conduction
studies showed an axonal sensory peripheral
neuropathy in the lower limbs. MRI revealed a
and punctate high T2 signal changes in the thalami
and heads of caudate nuclei. All lesions had high
ADC. MRI of the spinal cord was normal. The
patient declined skeletal muscle biopsy.
Both patients were compound heterozygous for
(p.T251I), c.1760 (p.P587L) and c.2243G>C
(p.W748S) mutations. B1 had the c.2209G>C
(p.737R) and c.2243G>C (p.W748S) mutations.
Figure 1. Imaging. Axial T2-weighted magnetic resonance
imaging showing diffuse leucoencephalopathy in patient A1.
mtDNA depletion in PEO
No other mutations were found. Sequencing of the
son of B1 confirmed that her mutations were in
trans. The sister of A1 had the T251I and P587L,
but not the W748S, suggesting that the T251I and
P587L were on the same allele, and in patient A1 in
trans with the W748S.
LPCR showed a typical pattern of multiple
mtDNA deletions in the skeletal muscle of patient
A1 (Fig. 2). Real-time PCR showed a reduced
ND1 ⁄ND4 ratio in the patient?s muscle (average of
two runs was 0.77) as compared with the controls,
suggesting that approximately 23% of the patient?s
mtDNA molecules harboured deletions affecting
the ND4 region (Fig. 3B). The ND1 ⁄18SrRNA
ratio was significantly lower than that of the
controls, suggesting an approximately 64% deple-
tion of total mtDNA in the patient?s skeletal
muscle (Fig. 3B).
We report two patients with late onset PEO plus
syndromes caused by mutations in POLG1. Each
patient carried different mutations (compound
heterozygotes) and while all mutations have been
reported previously, the combinations seen here are
novel. In addition, we demonstrate that mtDNA
depletion occurs in POLG1-associated PEO.
The G737R mutation (patient B1) has been
reported with other mutations giving either PEO
with neuropathy, Alpers? syndrome or a Charcot–
Marie–Tooth-like disease (1, 13, 15, 16). It has also
been reported as a single heterozygous mutation in
patients with early onset encephalopathy, but its
significance in these cases was unclear (16). The
W748S is one of the commonest POLG1 mutations
1:125 in Finland (18), and is mainly associated with
MSCAE and Alpers? syndrome (5–7). The T251I is
another common POLG1 mutation with a reported
carrier frequency of 1:90 in the Italian population
(1). It is most often found together with the P587L
on the same allele. The T251I is reported as a
homozygous mutation in arPEO and, in trans with
other mutations, giving arPEO and Alpers? syn-
drome (1, 7, 10, 16). The P587L usually co-
segregates on the same allele with the T251I with
the exception of four reported cases. It was found
with the P589L in cis and in trans with the W748S in
Figure 2. Long PCR results from skeletal muscle DNA. Lanes
1 and 6, 1 kb ladder (Promega; 250 b to 10 kb); lanes 2, 3, 4,
10 kb PCR; lane 2, patient A1; lane 3, control 1; lane 4, control
2; lanes 7, 8, 9, 15 kb product; lane 7, patient A1; lanes 8 and 9,
controls; lanes 5 and 10, negative controls (no template). In the
10 kb amplification, multiple smaller species are visible in the
patient?s mitochondrial (mt)DNA while in the 15 kb PCR, a
smaller single species of mtDNA is selectively amplified in the
patient?s muscle mtDNA.
Figure 3. Real-time PCR analysis in skeletal muscle DNA.
(A) Quantification of mitochondrial (mt)DNA. ND1 and ND4
mtDNA regions are quantified relative to nuclear DNA
(18SrRNA). The patient (arrow) shows depletion of both ND1
and ND4 compared with the two healthy controls (C1 and C4).
ND4 depletion seems more pronounced because of deletions in
that area. (B) Multiple deletions. Comparison of ND1–ND4
shows approximately 23% loss of ND4 relative to ND1
supporting the long PCR findings of multiple deletions.
Tzoulis et al.
a case of epilepsy (8), in trans with the R853W in a
case of PEO (19) and was reported as a single
heterozygous change in two patients with complex
PEO phenotypes (12). Its pathogenic role as a single
change, however, remains unclear. The sister of A1
had a mild ptosis phenotype suggesting that the
T251I_P587L alone may be pathogenic. No muscle
The molecular pathogenesis of PEO caused by
POLG mutations is poorly understood. At the
molecular level, this condition is consistently char-
acterized by multiple mtDNA deletions in the
skeletal muscle (1, 9–13). The mechanism by which
these deletions are generated and how they con-
tribute to the disease remains unclear, however, but
it is possible that mtDNA deletions cause respira-
tory chain impairment and loss of ATP produc-
tion. We found multiple mtDNA deletions in
muscle from patient A1 using both LPCR and
real-time PCR; real-time studies estimated that
approximately 23% of mtDNA molecules were
affected. In addition, real-time PCR demonstrated
a quantitative loss of mtDNA, with approximately
64% depletion of MtDNA. mtDNA depletion is a
common finding in the muscle and liver of patients
with severe POLG phenotypes such as Alpers?
syndrome (7, 8), and has been shown in some
fibroblast lines from such patient; it has not, to the
best of our knowledge, been reported in skeletal
muscle of patients with POLG-associated PEO
(11). In our case, the levels of depletion were
significantly higher than the levels of deletion. It is
therefore possible that, in addition to multiple
deletions, quantitative loss of the mitochondrial
genome also plays a major role in the pathogenesis
of PEO caused by POLG mutations.
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