Ophthalmoplegia Diagnosis
This is a type I deletion, which is caused by a
Dear Editor:
Chronic progressive external ophthalmoplegia (CPEO) fea-
tures insidious development of progressive external oph-
thalmoplegia and ptosis. The diagnosis of CPEO is based on
the results of a muscle biopsy including ragged-red fibers1,2
or by Southern blotting to verify alterations of mitochon-
drial DNA (mtDNA) in the skeletal muscle.1,2 However,
such deletions may not be found in leukocytes; therefore,
the diagnosis requires an invasive procedure to obtain the
skeletal muscle. We investigated the efficacy of long-range
polymerase chain reaction (PCR) to diagnose CPEO
mtDNA deletions in buccal cells and urinary epithelium of
2 patients with confirmed CPEO mtDNA deletions in me-
dial rectus muscle.
A 19-year-old female presented with complaints of pro-
gressive ptosis and an increasing inability to move her eyes
noted for 8 years. Bilateral complete ptosis with bilateral
total external ophthalmoplegia was present (Fig 1A [avail-
able at http://aaojournal.org]). She had 12 prism diopters
(PD) of exotropia at distance and near in the primary posi-
tion. She underwent unilateral medial rectus resection and a
frontalis sling with silicone rods.
A 23-year-old female presented with complaints of pro-
gressive ptosis for 9 years and an increasing inability to
move her eyes, noted for 3 years. Bilateral complete ptosis
with bilateral total external ophthalmoplegia was present
(Fig 1B [available at http://aaojournal.org]). She had exo-
tropia of 40 PD and a right hypertropia of 8 PD at distance
and an exotropia of 50 PD and right hypertropia of 6 PD at
near in the primary position. She underwent bilateral medial
rectus resection with an adjustable suture and frontalis sling
with silicone rods.
Total DNA was extracted from peripheral blood and
medial rectus muscle, hair follicles, urinary epithelium, and
buccal mucosa using the PureGene DNA isolation kit (Gen-
tra System Inc., Minneapolis, MN). Long-range PCR was
performed using Dr. Coulter-Mackie’s protocol with mod-
ification3 and the Expand Long Template PCR system
(Roche Applied Science, Mannheim, Germany). If the
long-range PCR revealed a deletion of mtDNA, the de-
letion region was narrowed down by restriction enzyme
mapping and the breakpoint was identified by gap PCR
and sequencing.
Case 1 showed a 4976-base pair (bp) deletion in the
muscle from nucleotide positions (np) 8470 to np 13446 of
the mtDNA.
13-bp repeat element (ACCTCCCTCACCA) flanking the
deletion breakpoints (Fig 2A [available at http://aaojournal.
org]). Case 2 had a 6333-bp deletion in the muscle from np
9650 to np 15982 of the mtDNA (Fig 2B [available at
http://aaojournal.org]). The deletion detected in the muscle
DNA was also identified in other tissue DNAs: buccal cells,
urine, skin, conjunctiva, and hair roots in Case 1, and in the
urine and buccal cells in Case 2. However, the DNA dele-
tion was not detected in the blood of either patient. Semi-
quantification results using densitometry appear in Fig 2
(available at http://aaojournal.org).
The mtDNA mutations associated with CPEO can be
categorized as follows: first, about 47% of patients with
CPEO have been reported to have a large, single deletion in
mtDNA that is not inherited.1 Second, many patients with-
out gross structural abnormalities of mtDNA have a point
mutation at many different nucleotide positions and dem-
onstrate a maternal pattern of inheritance.4 Third, a few
patients have multiple deletions and have an autosomal
dominant or recessive pattern of inheritance.
The molecular anlaysis of mtDNA in blood cells has not
identified a deletion in CPEO patients.1 Therefore, skeletal
muscle tissue was used for molecular diagnosis of CPEO.
Although this method is confirmatory, there are some draw-
backs, including the invasiveness of a surgical procedure to
obtain the skeletal muscle tissue, postoperative care, scar-
ring, and the handling of the muscle sample. Alternatively,
DNA for a genetic diagnosis can potentially be derived from
blood, hair roots, buccal mucosa, and urine. Herein, we
confirm that the mtDNA deletion identified in muscle was
also present in the DNA from buccal cells, urinary epithe-
lium, and hair roots by PCR. The quality of DNA isolated
from the buccal cells and urinary epithelium was sufficient to
amplify mtDNA. Therefore, our findings suggest that reliable
molecular diagnosis with PCR can be obtained on buccal cells
and urinary epithelium for the diagnosis of CPEO. In conclu-
sion, PCR of buccal cells and urinary epithelium could be a
less invasive method for the diagnosis of CPEO.
Acknowledgement. Supported by a grant of the Korea
Health 21 R&D Project, Ministry of health, welfare and
family affairs, Republic of Korea (A080299).
JEONG-MIN HWANG, MD
HO KYUNG CHOUNG, MD
HYUN SOO KO, MS
MOON-WOO SEONG, MD
JI YEON KIM, MD
SUNG SUP PARK, MD
Seoul, Korea
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 Ophthalmology Volume 116, Number 4, April 2009
References
1. Johns DR. Seminars in medicine of the Beth Israel Hospital,
Boston. Mitochondrial DNA and disease. N Engl J Med 1995;
333:638 – 44.
2. Schoser BG, Pongratz D. Extraocular mitochondrial myopa-
thies and their differential diagnoses. Strabismus 2006;14:
107–13.
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3. Coulter-Mackie MB, Applegarth DA, Toone JR, Gagnier L.
A protocol for detection of mitochondrial DNA deletions:
characterization of a novel deletion. Clin Biochem 1998;
31:627–32.
4. Mariotti C, Savarese N, Suomalainen A, et al. Genotype to
phenotype correlations in mitochondrial encephalomyopathies
associated with the A3243G mutation of mitochondrial DNA.
J Neurol 1995;242:304 –12.
 Letters to the Editor

Figure 1. Clinical photograph of the patient showing bilateral ptosis with total external ophthalmoplegia. A, Case 1; B, Case 2.

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Figure 2. Molecular genetic analyses of CPEO patients. A, Case 1. (1) Long-range PCR showed about 5-kb deletion of mtDNA. (2) Gap PCR and
sequencing revealed exact deletion range (np 8470-13446). (3) mtDNA deletion was also detected in buccal cells, urinary epithelium, and other tissues.
B, Case 2. (1) 6.5-kb mtDNA deletion. (2) Exact deletion range (np 9650-15982). (3) mtDNA deletion was also detected in buccal cells and urinary
epithelium. C ⫽ CPEO Case; CPEO ⫽ chronic progressive external ophthalmoplegia; M ⫽ molecular marker; N ⫽ normal control; np ⫽ nucleotide
positions; PCR ⫽ polymerase chain reaction.

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