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A Novel Gly35Ser Mutation in the RDH5 Gene in a Japanese Family With Fundus Albipunctatus Associated With Cone Dystrophy
Yuko Wada, MD;
Toshiaki Abe, MD;
Hajime Sato, MD;
Makoto Tamai, MD
Arch Ophthalmol. 2001;119:1059-1063.
ABSTRACT
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Objective To assess the clinical and genetic characteristics of a Japanese family
with fundus albipunctatus with progressive cone dystrophy associated with
a mutation in the RDH5 gene.
Design Case report with clinical findings and results of fluorescein angiography,
electroretinograms, kinetic visual field testing, dark adaptometry, and DNA
analysis.
Setting University medical center.
Patients We studied the ocular findings in 6 members of a Japanese family with
fundus albipunctatus with cone dystrophy and a guanine-to-adenine transversion
at the first nucleotide in codon 35 of the RDH5 gene.
The mutation resulted in a substitution of serine for glycine in amino acid
35 (Gly35Ser) of the RDH5 gene.
Results Characteristic features included poor night vision, white dots in the
retina, cone dystrophy, and a mottled appearance of the retinal pigment epithelium.
Electroretinograms showed greater impairment of the rod-mediated responses
than the cone-mediated responses. After 3 hours of dark adaptation, the a
and b waves and scotopic b waves recovered.
Conclusions Although the mutation of the RDH5 gene has
been known as a causative gene of fundus albipunctatus, the Gly35Ser mutation
in the RDH5 gene may be related to the pathogenesis
of progressive retinal degeneration. This phenomenon may provide evidence
of gene phenotype caused by a mutation in the RDH5
gene.
Clinical Relevance The Gly35Ser mutation causes fundus albipunctatus with cone dystrophy.
This finding provides evidence that some kinds of mutations in the RDH5 gene are related, in part at least, to the pathogenesis of progressive
retinal degeneration.
INTRODUCTION
FUNDUS ALBIPUNCTATUS is a rare form of congenital stationary night blindness
that is characterized by scattered white dots in the fundus and extremely
slow dark adaptation of the rod photoreceptors. This disease is inherited
as an autosomal recessive trait, and most patients with fundus albipunctatus
show poor night vision.
In 1999, the RDH5 gene, which encodes 11-cis-retinol dehydrogenase, was reported to be a causative
gene for fundus albipunctatus.1 It is well
known that 11-cis-retinol dehydrogenase catalyzes
the 11-cis-retinol to 11-cis-retinal
reaction. To date, 2 kinds of mutation, designated as Gly238Trp and Ser73Phe
mutations, have been reported.1 Earlier, we
identified a common Leu310 (1–base pair [bp] deletion [del]; +4-bp insertion
[ins]) (CTT to GAAGTT) mutation in the RDH5 gene
in 4 unrelated Japanese families with fundus albipunctatus.2
Patients with this mutation in the RDH5 gene showed
scattered white dots in the retina and poor night vision. Fluorescein angiography
disclosed irregular hyperfluorescent areas mainly in the posterior portion
in all patients. These results demonstrated that the RDH5 gene mutation caused the impairment of the retinal pigment epithelium
and suggested the possibility that mutations in the RDH5 gene were related not only to fundus albipunctatus but also to progressive
retinal degeneration. In addition, fundus albipunctatus associated with macular
degeneration has been reported.3-5
Thereafter, we screened 200 unrelated patients with autosomal recessive
retinitis pigmentosa and 1 patient with fundus albipunctatus associated with
cone dystophy to search for mutations in the RDH5
gene. In this report, we describe the ocular findings associated with a newly
identified RDH5 mutation in a Japanese family with
fundus albipunctatus associated with cone dystophy. The aim of this study
was to assess the phenotypic manifestations associated with this molecular
alteration. We shall show that the mutation gave rise to a guanine-to-adenine
transversion in the first nucleotide at codon 35, resulting in substitution
of a serine residue for a glycine residue (Gly35Ser). The clinical features
associated with this mutation disclosed a degenerative macula in the right
eye and chorioretinal atrophy in the left eye with scattered white dots in
the retina. Eight years of observation disclosed a progressive decrease in
visual acuity, an enlargement of the macular degeneration, and attenuation
of the retinal vessels. These observations make up the natural course of the
phenotype associated with the Gly35Ser mutation in the RDH5 gene.
PATIENTS AND METHODS
We screened 200 genomic DNA samples isolated from unrelated patients
with autosomal recessive retinitis pigmentosa and 1 patient with fundus albipunctatus
associated with cone dystrophy to search for mutations in the RDH5 gene. Informed consent was obtained from all patients before their
entry into this study. Mutation screening was performed by the polymerase
chain reaction followed by nonradioisotopic single-strand conformation polymorphism
as previously reported.6 The amplified DNA
fragment then underwent electrophoresis in 8% nondenaturing polyacrylamide
gel containing 10% glycerol at 20 W for 8 hours at room temperature. The DNA
bands were visualized by silver staining. Subsequently, amplified DNA fragments
that showed abnormal band shift on a single-strand conformation polymorphism
gel were directly sequenced (ABI sequencer; Applied Biosystems, PerkinElmer,
Inc, Boston, Mass).
One pedigree with fundus albipunctatus associated with cone dystrophy
was included in this study (Figure 1).
A homozygous Gly35Ser mutation in the RDH5 gene was
identified in 1 affected member, and the parents were found to be heterozygous
for the Gly35Ser mutation. In addition, we screened the peripherin/RDS, ROM1, rhodopsin, and RLBP1 genes to search for other mutations in the families.
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Figure 1. Pedigree of a Japanese family
with fundus albipunctatus with cone dystrophy showing affected (solid symbols)
and unaffected (open symbols) members. Squares indicate male members; circles,
female members; X, individuals examined in this study; slash, deceased; dot
in the circle or in the square, carrier; arrow, proband; double horizontal
line, consanguineous marriage; M, mutant allele; and plus sign, normal allelle.
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The phenotypic features of the patient who showed the Gly35Ser mutation
were studied to characterize the mutation. The ophthalmic examination included
best-corrected visual acuity, kinetic visual field examination, slitlamp biomicroscopy,
fundus examination, fluorescein angiography, and electroretinograms (ERGs).
Kinetic visual field examination was performed by Goldmann perimetry with
V-4-e and I-4-e isopters.
The ERG recordings were performed under controlled conditions as reported
previously and conformed to the International Society for Clinical Electrophysiology
of Vision standards.7 Briefly, ERGs were obtained
with a single flash or a 30-Hz flicker stimulus of red light under light-adapted
conditions for cone-isolated responses, a dim blue flash in the dark-adapted
condition (30 minutes) for rod-isolated responses, and a white standard flash
(intensity of 1.69 candela/m2 per second) in the dark-adapted condition
for eliciting maximal responses of rods and cones. Ganzfeld dark-adapted thresholds
were recorded with the use of a Goldmann-Weekers dark adaptometer (Haag-Streit
AG, Liebefeld, Switzerland). Color vision was tested with the D-15 panel.
RESULTS
DNA ANALYSIS
One affected member (Figure 1)
showed an abnormal band shift on a single-strand conformation polymorphism
gel, and the abnormal band shift cosegregated with the disease. The obligate
carriers (I:1 and I:2) showed both abnormal and normal bands. The abnormal
nucleotide sequence was a homozygous guanine-to-adenine transversion at the
first nucleotide of codon 35. This alteration caused a serine substitution
for a glycine residue in codon 35 of the RDH5 gene
and was designated as the Gly35Ser mutation (Figure 2). The carriers were heterozygous for this mutation, and
the nonaffected members did not show this mutation. We further screened our
patients to search for mutations in the peripherin/RDS, ROM1, rhodopsin, and RLBP1 genes,
and none was found. We confirmed that the Gly35Ser mutation cosegregated with
the disease and was not detected in the other 200 patients with autosomal
recessive retinitis pigmentosa or 100 normal control subjects.
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Figure 2. Results of nucleotide sequencing
analysis. The upper sequence, a normal allele from a normal subject, shows
GCC in codon 35. The middle sequence from the carrier (I:1) shows both normal
and mutant alleles. Arrows indicate the position of the heterozygous mutation
(G/A). The bottom sequence from patient II:1 shows a homozygous AGC in codon
35, resulting in the substitution of serine for glycine.
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REPORT OF A CASE
A 51-year-old man had initially noticed poor night vision during his
early teens and was diagnosed as having fundus albipunctatus by a local ophthalmologist.
His parents were first cousins. The patient experienced a gradual progression
of visual impairment, including constriction of the visual field, night blindness,
and photophobia. In 1992, at 41 years of age, he visited our clinic to have
a detailed assessment of his eyes. The visual acuity was corrected to 20/50
OD with a +0.25 -0.5 x 40 refraction and 20/20 OS with a refraction
of -0.75 x 80 cyl.
Slitlamp biomicroscopic examination showed normal-appearing cornea,
anterior chamber, iris, lens, and vitreous in both eyes. Fundus examination
showed atrophic macular lesions in the right eye and 3 sharply demarcated
macular lesions in the left eye with scattered white dots in the retina. The
retinal vessels were attenuated (Figure 3). Fluorescein angiography demonstrated hypofluorescent areas, indicating
chorioretinal atrophy in the macula of the left eye and granular hyperfluorescent
areas mainly around the macula in the right eye. Irregular hyperfluorescent
spots were seen in the central and midperipheral regions bilaterally (Figure 4). Kinetic visual field testing showed
ring scotomas bilaterally (Figure 5).
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Figure 3. Fundus photographs. A, Fundus
photograph of patient II:1 at age 43 years. Scattered white dots and macular
degeneration are observed in the right eye. B, Three sharply demarcated chorioretinal
lesions are seen in the macula of the left eye. C, At age 51 years, the retinal
degeneration and attenuation of retinal vessels have progressed. D, Enlargement
of the chorioretinal atrophy, which is pigmented in the left eye.
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Figure 4. A, Fluorescein angiogram of patient
II:1. Granular hyperfluorescence is observed around the macular regions and
in the midperipheral area of the right eye. B, Hypofluorescent areas caused
by chorioretinal atrophy are seen in the macular and nasal regions of the
left eye. Diffuse hyperfluorescence in the midperipheral and attenuated retinal
vessels is seen.
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Figure 5. Results of Goldmann visual field
testing demonstrating a ring scotoma in both eyes with preserved peripheral
visual field.
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After 30 minutes of dark adaptation, rod b waves and cone b waves were
unrecordable, while the 30-Hz flicker responses were extremely reduced (42.6
µV in the right eye and 21.3 µV in the left eye) with delayed
implicit times. The normal range in our laboratory is 83 to 175 µV.
The standard flash ERG disclosed severely decreased a and b waves in both
eyes. After 3 hours of dark adaptation, the standard flash ERG and scotopic
ERG showed recovery of the a and b waves and scotopic b waves, respectively,
although they did not recover to the normal level. The cone b waves showed
no significant change after 3 hours of dark adaptation. Blink artifacts were
seen in the 3-hour dark-adapted scotopic and photopic ERGs (Figure 6). The patient could not discriminate colors. The dark-adaptation
curve was 1.0 log unit above normal threshold after 1 hour of adaptation but
attained normal threshold values after 3 hours in the dark.
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Figure 6. Electroretinograms of patients
I:1, I:2, and II:1. The rod-isolated responses of patient II:1 are nonrecordable.
After 30 minutes of dark adaptation, the standard electroretinogram showed
reduced a and b waves. After 3 hours of dark adaptation, the amplitudes of
a and b waves and scotopic b waves have recovered. The amplitude of 30-Hz
flicker responses was reduced. After 30 minutes of dark adaptation, the standard
electroretinograms of patients I:1 and I:2 are of normal amplitude. Del indicates
deletion; ins, insertion.
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The patient experienced accelerated deterioration of night vision and
central visual acuity after his first visit in 1992. Visual acuity decreased
to 20/200 OD and 20/250 OS at the age of 50 years. Fundus examination at this
time disclosed bilateral atrophic macular degeneration, which appeared to
have progressed since his first visit, and diffuse retinal mottling in the
midperipheral areas. The retinal vessels appeared more attenuated (Figure 3). His parents had no visual symptoms,
and slitlamp biomicroscopy of his parents showed normal-appearing cornea,
anterior chamber, iris, lens, and vitreous; fundus examination showed no retinal
changes in both eyes; and the standard flash ERGs were normal.
COMMENT
It is well known that a deficiency of vitamin A causes impaired night
vision and retinal degeneration. Thus, it has been thought that genes related
to visual cycle or vitamin A metabolism were candidate genes for retinal degeneration
or night blindness.
In 1999, it was reported that fundus albipunctatus was caused by mutations
of the RDH5 gene, which is localized on chromosome
12q13-q14 and encodes 11-cis-retinol dehydrogenase.1 This enzyme is abundantly expressed in the retinal
pigment epithelium and is a 32-kd membrane-bound enzyme with 318 amino acids.
It catalyzes the conversion of 11-cis-retinol to
11-cis-retinal and plays an important role in the
synthesis of functional visual pigment.
A previous study by our group showed that a Leu310 (1-bp del; +4-bp
ins) (CTT to GAAGTT) mutation in the RDH5 gene was
found as a frequent cause of fundus albipunctatus in the Japanese population.2 Moreover, we found slight variations in the clinical
features of patients with this mutation. In particular, the presence of irregular
hyperfluorescent spots on fluorescein angiography led us to suggest that the RDH5 gene might also be a candidate gene for progressive
retinal degeneration. We therefore extended our study and identified a new
Gly35Ser mutation in a previously unidentified patient with fundus albipunctatus
with cone dystrophy.
As for the Gly35Ser mutation, Gly35 is the first sequence G-X-X-X-G-X-G,
which is a highly conserved cofactor binding motif. Because Gly35 is considered
to be at an important position, cases with a mutation at codon 35 may show
more severe changes than those with other mutations in the RDH5 gene.8
The clinical features produced by this mutation were decreased visual
acuity, night blindness, white dots in the retina, progressive retinal degeneration,
and attenuated retinal vessels. Furthermore, the ERGs showed that the recovery
of rod function was abnormally delayed and cone responses were severely impaired.
These features were different from those of patients associated with the Leu310
(1-bp del; +4-bp ins) (CTT to GAAGTT) mutation except for scattered white
dots and night blindness. This mutation was not found in 200 patients with
autosomal recessive retinitis pigmentosa.
Taken together with our previous results,2
the clinical expressions associated with the RDH5
mutation varied from fundus albipunctatus alone to fundus albipunctatus with
cone dystrophy. It has been suggested that there may be a spectrum of gene
phenotypes caused by mutations in the RDH5 gene.
Mutations in the rhodopsin and BPDE genes have
been implicated not only in congenital stationary night blindness but also
in retinitis pigmentosa.9-12
These findings can then support the earlier suggestion that mutations in the RDH5 gene are responsible not only for fundus albipunctatus
but also for progressive retinal degeneration. However, they cannot explain
the variable expressivity associated with the RDH5
gene mutation. As in the digenic inheritance pattern of retinitis pigmentosa
caused by mutations in both the peripherin/RDS and ROM1 genes,13 there may
be other mutations in the RDH5 Gly35Ser mutation
that cause the cone dystrophy.
To examine this possibility, we also screened other candidate genes,
such as rhodopsin, peripherin/RDS, ROM1, and RLBP1, to search for additional
mutations. The results did not disclose any disease-causing mutation in these
genes, and, therefore, mutations in at least these 4 genes are unlikely to
be related to the pathogenesis of fundus albipunctatus with cone dystrophy
in our patient. However, further molecular genetic analysis is needed to eliminate
this possibility.
In conclusion, we identified a homozygous Gly35Ser mutation in the RDH5 gene in a Japanese patient with fundus albipunctatus
associated with cone dystrophy. This finding provides evidence that a mutation
in the RDH5 gene is related, at least in part, to
the pathogenesis of progressive retinal degeneration.
AUTHOR INFORMATION
Accepted for publication December 28, 2000.
This study was supported in part by a grant from the Research Committee
on Chorioretinal Degenerations and Optic Atrophy, Ministry of Health and Welfare
of the Japanese Government, Tokyo, Japan (Dr Tamai), and grant-in-aid for
scientific research A-2-10307041 from the Ministry of Education, Science,
and Culture of the Japanese Government, Tokyo (Dr Tamai).
Corresponding author and reprints: Yuko Wada, MD, Department of Ophthalmology,
Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-77,
Japan (e-mail: yukow{at}oph.med.tohoku.ac.jp).
From the Department of Ophthalmology, Tohoku University School of Medicine,
Sendai, Japan.
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