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Corneal Endothelial Degeneration in Dentatorubral-Pallidoluysian Atrophy
Daisuke Ito, MD;
Masakazu Yamada, MD;
Masataka Kawai, MD;
Tomohiko Usui, MD;
Junnich Hamada, MD;
Yasuo Fukuuchi, MD
Arch Neurol. 2002;59:289-291.
ABSTRACT
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Background Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant
spinocerebellar degeneration that exhibits a variety of neurologic manifestations.
However, only a few reports have studied disturbances outside the central
nervous system. We described 2 unrelated patients with DRPLA accompanied by
corneal endothelial degeneration.
Patients and Methods A 52-year-old man presented with cerebellar ataxia and dementia. Magnetic
resonance imaging of the brain showed cerebellar atrophy. Dentatorubral-pallidoluysian
atrophy was diagnosed because of the detection of expansion of CAG repeats
at the DRPLA locus. On admission, his visual acuity
was severely impaired. Specular microscopy showed decreased endothelial cell
density (500 cells/mm2) compared with that of healthy subjects.
The second patient was a 69-year-old man with cerebellar ataxia. Magnetic
resonance imaging of the brain showed cerebellar and brainstem atrophy. The
diagnosis of DRPLA was based on expanded CAG repeats of the DRPLA gene. Specular microscopy showed significant decrease of endothelial
cell density (1506 cells/mm2). Reverse transcriptase–polymerase
chain reaction analysis showed DRPLA gene expression
in corneal endothelial cells.
Conclusions Mutant DRPLA protein may be directly associated with corneal endothelial
degeneration. Corneal endothelial cell loss is an important sign of DRPLA,
and the corneas of patients with DRPLA should be examined.
INTRODUCTION
DENTATORUBRAL-pallidoluysian atrophy (DRPLA) is a member of the family
of autosomal dominant cerebellar ataxias. Patients may have a variety of symptoms,
including myoclonus, seizures, choreiform movements, cerebellar ataxia, dementia,
and psychiatric disturbance.1 Only a few reports
have studied disturbance of the extracentral nervous system in autosomal dominant
cerebellar ataxia. Recently, Abe et al2-3
reported corneal endothelial changes in patients with spinocerebellar ataxia
type 1 (SCA1) and DRPLA, and suggested a significant correlation between decreased
corneal endothelial cell density and autosomal dominant cerebellar ataxias.
In the present study, we report 2 cases of DRPLA accompanied by corneal endothelial
cell loss and the expression of the DRPLA gene in
corneal endothelial cells in unrelated patients and discuss the molecular
pathogenesis of corneal endothelial degeneration in DRPLA.
REPORT OF CASES
CASE 1
A 52-year-old Japanese man had been well until 49 years of age. His
grandfather had died of cerebellar ataxia with severe visual disturbance (a
genetic analysis was not performed). The patient first noticed gait disturbance
and dysarthria at 50 years of age, and these symptoms gradually progressed
for 2 years. At 52 years of age, he was admitted to Keio University Hospital,
Tokyo, Japan, for the first time. The results of a neurologic examination
showed dysarthria, ataxic gait, disturbance of coordination in the upper and
lower limbs, pyramidal tract signs, and dementia. Magnetic resonance imaging
of the brain showed cerebellar atrophy and dilation of the cerebral ventricles.
Electromyographic studies demonstrated slightly low conduction velocity of
the motor nerve. Genetic analysis showed that the expanded allele had 62 CAG
repeats at the DRPLA locus, whereas the normal allele
contained 18 repeats.
At admission to our hospital, he complained of visual disturbance. His
best corrected visual acuity was 20/25 in his right eye and 20/70 in his left
eye. An examination of the pupils, ocular media, and fundus showed normal
findings, but severe nystagmus was observed. The results of slitlamp examination
showed corneal edema and guttae, which are excrescences of the Descemet membrane
produced by abnormal endothelial cells. On specular microscopy, the corneal
endothelial cells were enlarged, and the endothelial cell density (500 cells/mm2 OD and 500 cells/mm2 OS) was severely decreased compared
with that of age-matched healthy subjects (mean [± SD] for age-matched
healthy subjects, 2685 ± 94 cells/mm2).4
CASE 2
A 69-year-old Japanese man first noticed gait disturbance at 61 years
of age. His father and elder sister had similar cerebellar ataxias (genetic
analysis was not performed). He visited our hospital at 66 years of age because
of dysarthria and worsening of his gait disturbance. The results of a neurologic
examination showed dysarthria, ataxic gait, and disturbance of coordination
in the upper and lower limbs. Magnetic resonance imaging of the brain showed
cerebellar and brainstem atrophy. An analysis of DNA revealed that he had
an expanded allele of the DRPLA gene with a normal
allele (expanded/normal CAG repeats, 56/11).
At 63 years of age, he had keratitis due to herpes simplex and corneal
transplantation in his right eye, but his left eye was not affected then.
At 69 years of age, his corrected visual acuity was 20/20 OS. On specular
microscopy, the endothelial cell density of the cornea in his left eye (1506
cells/mm2 OS) was significantly decreased compared with that of
healthy subjects (mean [± SD] for age-matched healthy subjects, 2711
± 121 cells/mm2) (Figure
1).4 An examination of the pupils,
ocular media, and fundus and electroretinography showed normal findings.
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Figure 1. Corneal endothelial cells of patients
with dentatorubral-pallidoluysian atrophy and spinocerebellar ataxia type
6 (SCA6) visualized under specular microscopy. A, Case 2 (1506 cells/mm2). B, A patient with SCA6 (3300 cells/mm2). The corneal
endothelial cell density of case 2 was severely decreased compared with that
of the patient with SCA6.
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DETECTION OF DRPLA GENE EXPRESSION IN CORNEAL
ENDOTHELIAL CELLS
To study DRPLA gene expression in human corneal
endothelial cells, reverse transcriptase–polymerase chain reaction (RT-PCR)
analysis was performed. We produced complementary DNA (cDNA) of the human
corneal endothelial cell from human corneal endothelial cells, which were
isolated from healthy donor cornea (obtained from Rocky Mountain Lions Eye
Bank, Denver, Colo) as described previously.5
Total RNA was first isolated from human corneal endothelial cells using a
monophasic solution of phenol and guanidine isothiocyanate (Isogen; Nippon
Gene, Toyama, Japan), then treated with RNase-free DNase I (Stratagene, La
Jolla, Calif) for 30 minutes. Human brain cDNA was purchased (Maxim Biotech,
Inc, San Francisco, Calif), and amplification of the 557-base pair fragment
of the DRPLA gene, which is designed to amplify exons
9 to 10, was performed using the 5' primer 5'-AGGAGGACTACTACAGTCAC-3'
and 3' primer 5'-TGGTTTTGGTTGGGATGTTT-3'. For the negative
control of corneal endothelial cells, PCR was performed in the absence of
RT. The PCR buffer contained 1.5mM magnesium chloride with 0.2mM of each deoxynucleotide
triphosphate, 2µM of each primer, and 2.5 U of Taq polymerase (Takara Shuzo Co, Ltd, Shiga, Japan). The PCR analysis
consisted of 1 cycle of 5 minutes at 94°C; 35 cycles of 1 minute at 94°C,
1 minute at 60°C, and 2 minutes at 72°C; and 1 cycle of 7 minutes
at 72°C in a PCR system (GeneAmp System 2400; Perkin Elmer, Foster City,
Calif). The PCR products were separated by means of electrophoresis through
an 8% polyacrylamide gel and visualized using ethidium bromide under UV light.
As shown in Figure 2, RT-PCR
of human brain and corneal endothelial cells yielded the amplified band at
the expected size for the DRPLA gene. This PCR product
was also confirmed by restriction of the fragment length using MvaI and HhaI (New England Biolabs, Beverly,
Mass) (data not shown). This finding demonstrates that the DRPLA gene is expressed in corneal endothelial cells and brain.
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Figure 2. Expression of dentatorubral-pallidoluysian
atrophy gene in human corneal endothelial cells examined by means of reverse
transcriptase–polymerase chain reaction analysis. Lane M indicates molecular
marker; lane 1, human brain; lane 2, human corneal endothelial cells; and
lane 3, negative control without reverse transcriptase. The arrowhead indicates
the position of amplified DNA (557 base pairs [bp]).
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COMMENT
In the present study, we described 2 unrelated patients with DRPLA who
exhibited corneal endothelial cell loss but were free of other forms of ocular
changes. A decrease in endothelial cell density is generally caused by aging,
trauma, or inflammation. Case 1 had no history of ocular disease or trauma.
Although case 2 had a history of keratitis due to herpes simplex and of corneal
transplantation in his right eye, his left eye, which also showed a decrease
in endothelial cell density, had no such history. These decreases in cell
density were significant compared with those of age-matched healthy subjects.
The possibility of Fuchs endothelial dystrophy, which is characterized by
a decrease in endothelial cell density and corneal guttae, must also be considered
in these cases. Although the precise incidence of Fuchs endothelial dystrophy
in the general Japanese population is unclear, Fuchs dystrophy has been reported
to be extremely rare in Japan.6 Therefore,
it seems unlikely that Fuchs dystrophy would be present in the 2 unrelated
patients described herein. In addition, no corneal changes were detected in
patients with other autosomal dominant cerebellar ataxias (1 patient with
SCA2, 2 patients with Machado-Joseph disease, 1 patient with SCA6, and 2 patients
with unknown genetic loci) (Figure 1).
Recently, Abe et al2-3 also described
2 related patients with DRPLA who exhibited a reduction in corneal endothelial
cell density; corneal changes were not observed in the unaffected family members.
Our findings provide an independent confirmation of these studies and strongly
suggest that corneal endothelial cell loss is an important sign of DRPLA.
Another important finding of our report was that corneal endothelial
cells clearly express the DRPLA gene (Figure 2). Although the DRPLA gene is reported
to be widely expressed in various tissues, including unaffected organs, high
levels of expression are observed predominantly in neuronal tissue.7 Because corneal endothelial cells are derived from
the neuroectoderm,8 the presence of DRPLA gene expression in corneal endothelial cell is not surprising.
Recent investigations suggest that the aggregation of mutant proteins containing
expanded polyglutamine stretches plays an important role in neuronal degeneration.1 Our findings imply that the mutant DRPLA protein may
be directly associated with corneal endothelial degeneration and neuronal
degeneration.
Finally, further study is needed to elucidate whether the decrease in
corneal endothelial cell density is associated with the duration of the disease
and the number of expanded CAG repeats in the DRPLA
gene. Whether the nuclear aggregation of mutant DRPLA protein occurs in corneal
endothelial cells should also be investigated. In addition, we have evaluated
the corneas of only 2 Japanese patients with DRPLA. The occurrence of corneal
endothelial degeneration in patients with DRPLA should also be investigated
in other populations. Nevertheless, the present findings suggest that the
corneas of patients with DRPLA should be thoroughly examined.
AUTHOR INFORMATION
Accepted for publication September 18, 2001.
Author contributions: Study concept and design (Drs Ito, Hamada, and Fukuuchi); acquisition of data (Drs Ito, Yamada, Kawai, and Hamada); analysis and interpretation
of data (Drs Ito, Yamada, Kawai, and Usui); drafting
of the manuscript (Dr Ito); criticial revision of
the manuscript for important intellectual content (Drs Yamada,
Kawai, Usui, Hamada, and Fukuuchi); administrative, technical, and material
support (Drs Ito, Yamada, Kawai, and Usui); study supervision (Drs Hamada and Fukuuchi).
We thank Kinya Ishikawa, MD, and Hidehiro Mizusawa, MD, Tokyo Medical
and Dental University, for the genetic analysis in case 1, and Mamoru Shibata,
MD, Department of Neurology, Keio University, for the genetic analysis in
case 2.
Corresponding author and reprints: Daisuke Ito, MD, Department of
Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan (e-mail: di49{at}med.keio.ac.jp).
From the Departments of Neurology (Drs Ito, Hamada, and Fukuuchi) and
Ophthalmology (Drs Yamada and Kawai), School of Medicine, Keio University,
and the Department of Ophthalmology, Faculty of Medicine, University of Tokyo
(Dr Usui), Tokyo, Japan.
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gene. Br J Ophthalmol. 1999;83:124-125.
3. Abe T, Abe K, Aoki M, Itoyama Y, Tamai M. Ocular changes in patients with spinocerebellar degeneration and repeated
trinucleotide expansion of spinocerebellar ataxia type 1 gene. Arch Ophthalmol. 1997;115:231-236.
ABSTRACT
4. Yee RW, Matsuda M, Schultz RO, Edelhauser HF. Changes in the normal corneal endothelial cellular pattern as a function
of age. Curr Eye Res. 1985;4:671-678.
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6. Santo RM, Yamaguchi T, Kanai A, Okisaka S, Nakajima A. Clinical and histopathologic features of corneal dystrophies in Japan. Ophthalmology. 1995;102:557-567.
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7. Nagafuchi S, Yanagisawa H, Ohsaki E, et al. Structure and expression of the gene responsible for the triplet repeat
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