(Investigative Ophthalmology and Visual Science. 2000;41:3933-3935.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Mutations in the 11-cis Retinol Dehydrogenase Gene in Japanese Patients with Fundus Albipunctatus
Eri Hirose1,2,3,
Yumiko Inoue1,3,
Hiroyuki Morimura1,
Norio Okamoto2,
Masakatsu Fukuda2,
Shuji Yamamoto1,
Takashi Fujikado1 and
Yasuo Tano1
1 From the Department of Ophthalmology, Osaka University Medical School; and the
2 Department of Ophthalmology, NTT West Osaka Hospital, Japan.
 |
Abstract
|
|---|
PURPOSE. To detect mutations in the RDH5 gene encoding
11-cis retinol dehydrogenase in patients from Japan with
fundus albipunctatus.
METHODS. Polymerase chain reaction and direct genomic sequencing techniques were
used to detect mutations of the RDH5 coding exons (exons
2–5) in two unrelated patients with fundus albipunctatus. Selected
alleles that altered the coding region or intron splice sites were
evaluated further through segregation analysis in the families of the
index cases.
RESULTS. Two novel RDH5 mutations were identified. One of these
was a missense mutation Val264Gly in exon 5, and the other was an
in-frame insertion of 3 bp in exon 5.
CONCLUSIONS. The data indicate that mutations in RDH5 are the primary
cause of fundus albipunctatus.
|
Introduction
|
|---|
Fundus albipunctatus is a rare autosomal recessive form of
stationary night blindness characterized by numerous small, subretinal,
white or pale-yellow spots in the perimacula and the periphery of the
retina. This disease was first described by Lauber in 1910 who
distinguished it from an ophthalmoscopically similar disorder,
retinitis punctata albescens.1
Although young patients
with retinitis punctata albescens and fundus albipunctatus can have
similar symptoms, electroretinograms (ERGs), and fundus appearances,
patients with retinitis punctata albescens ultimately experience visual
field deficits and attenuated retinal vessels, whereas those with
fundus albipunctatus have a stationary condition. Mutations in the
genes encoding rhodopsin, peripherin/RDS, and cellular retinaldehyde
binding protein (CRALBP) have been reported in patients with retinitis
punctata albescens.2
3
4
5
6
The only reported gene to cause
fundus albipunctatus is that encoding 11-cis retinol
dehydrogenase with mutations being reported in only four families to
date.7
8
Among these four families, all members of
which were white and of European ancestry, four missense mutations
(Gly238Trp, Ser73Phe, Arg280His, and Ala294Pro) were found.
11-cis Retinol dehydrogenase is found in the retinal
pigment epithelium (RPE) and participates in the visual cycle.
11-cis Retinal is photoisomerized to all-trans
retinal, which is then reduced by the photoreceptor
all-trans retinal dehydrogenase.9
The
all-trans retinol produced is then transported to the RPE
where it is converted back to 11-cis retinal.10
11-cis Retinol dehydrogenase is abundant in the RPE, where
it converts 11-cis retinol to 11-cis
retinal.11
The enzymatic activities of two of the reported
mutant enzymes were strikingly reduced.7
|
Methods
|
|---|
This study conformed to the tenants of the Declaration of
Helsinki. We evaluated two patients from two families with fundus
albipunctatus and 50 normal control subjects. The patients had symptoms
of night blindness and multiple, subretinal, small white spots. There
were no visual field deficits. The ERGs were examined using white flash
(cone–rod response) or a 30-Hz red-flicker stimulus (cone-isolated
response) under adequate dark-adapted conditions (15 minutes or 120
minutes; portable ERG PE-300; Tomey, Waltham, MA). Fifty persons with
no known blood relative with hereditary retinal degeneration and no
symptoms of retinal malfunction served as control subjects. All
individuals resided in Japan. After informed consent was obtained, 10
to 20 ml venous blood was collected from each patient or control
subject and DNA extracted.
Exons 2 through 5 (the coding exons) of RDH5 were
individually amplified by polymerase chain reaction (PCR) using primer
pairs based on the published genomic sequence.7
12
Each
reaction used 50 to 100 ng DNA in 20 µl of a solution containing 20
mM Tris-HCl (pH 8.4); 0.25 to 1.5 mM MgCl2; 50 mM
KCl; 0.02 mM each of dTTP, dCTP, dGTP, and dATP; 0.1 mg/ml bovine serum
albumin (BSA); 0% or 10% dimethyl sulfoxide; and 0.25 U
Taq polymerase. The pH, Mg2+
concentration, and presence or absence of 10% dimethyl sulfoxide were
optimized for each primer pair. After initial denaturation (94°C for
5 minutes), 35 cycles of PCR were performed. Each cycle consisted of
denaturation (94°C for 30 seconds), primer annealing (56°C–60°C
for 30 seconds; exons 2A, 2B, 3A, 3B, 5A, and 5B at 60°C and exons 2C
and 4 at 56°C), and extension (71°C for 30 seconds). All exons in
all patients were evaluated by sequencing of PCR-amplified DNA segments
by means of a commercial sequencing protocol (Big Dye;
Perkin–Elmer/Applied Biosystems, Foster City, CA). Each sequence
variant expected to affect the protein sequence was further evaluated
by segregation analysis. For this purpose, DNA samples from relatives
were analyzed by direct genomic sequencing.
|
Results
|
|---|
Sequence analysis of RDH5 revealed homozygous mutations
in two index patients. One patient (P703) was homozygous for the
missense change Val264Gly (GTG
GGG; Fig. 1A
). This change was present homozygously in all three affected siblings
of the index patient (Fig. 2A
). The daughter of an affected sibling was a heterozygote. The second
index patient (P724) was homozygous for an in-frame mutation in codon
310 in exon 5 (Fig. 1B)
. The mutation replaces codon 310, which
normally specifies Leu, with two codons specifying Glu and Val
(Leu310GluVal; CTTGAAGTT). Only one unaffected sibling (homozygous
wild-type) was available for segregation analysis (Fig. 2B)
. Neither of
these mutations was found among a set of 50 Japanese control subjects.
View larger version (59K):
[in this window]
[in a new window]
|
Figure 1. DNA sequence of RDH5 mutations. (A) Sequence
surrounding codon 264 in P860, who was a sibling of P703, the proband.
The sequence of a normal control individual is shown for comparison.
(B) Sequence surrounding codon 310 in P724 and in a normal
control subject. Arrows: Mutation region.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. Pedigrees of two unrelated families with fundus albipunctatus.
(A) Family T had three affected siblings, all of whom
carried the Val264Gly mutation homozygously. The daughter of one
affected sibling was heterozygous. (B) Family K has only one
affected individual and one unaffected sibling. (Arrow
A, B) Proband.
|
|
Both index patients had normal visual acuity of 20/20 and full visual
fields. The fundi of P703 at age 45 and P724 at age 50 showed small
white deposits at the level of the RPE (Fig. 3)
. There was no attenuation of the retinal vessels. The ERGs showed a
reduction in the b-wave amplitude to white flash light after 15 minutes
of dark adaptation, which recovered to normal within 2 hours in P703
(Fig. 4)
.
View larger version (92K):
[in this window]
[in a new window]
|
Figure 3. Fundus photographs of the proband (P703) in family T (A) at
age 45 and the proband (P724) of family K (B) at age 55.
Both had light yellow spots in the periphery but not in the
macula within the vascular arcade. The retinal vessels appeared to be
of normal caliber.
|
|
View larger version (69K):
[in this window]
[in a new window]
|
Figure 4. Full-field ERGs of a P703. Recordings were made after white
flashes (cone–rod response; A, C) and during red
flicker (cone-isolated response; B, D).
Calibration bars are to the lower right of the waveforms.
The upper and lower waves in each panel
correspond to the right and left eye, respectively. The b-wave
amplitude (right eye, 316 µV; left eye, 340 µV) in the eye of P703
after 15 minutes of dark adaptation on white flashes was reduced
(A); however, the b-wave amplitude (C; right eye,
532 µV; left eye, 580 µV) of P703 after 120 minutes of dark
adaptation on white flashes recovered to normal control (right eye, 480
µV; left eye, 468 µV; E). Cone-isolated responses
(B, D) had almost no changes between 15 minutes
and 120 minutes of dark adaptation (right eye, 40.0–54.6 µV; left
eye, 60.0–76.0 µV). Normal amplitude is 53.3 µV in the right eye
and 62.6 µV in the left eye, respectively. White flashes
(E) and 30-Hz red flicker (F) in a normal control
subject.
|
|
|
Discussion
|
|---|
The RDH5 mutations found in this study are likely
to be pathogenic. Both mutations were present homozygously in affected
individuals, in agreement with the recessive inheritance pattern. In
both families the mutations cosegregated with the disease. Finally,
results showed that the mutation Val264Gly changes a nonpolar residue
to a polar one, and the mutation Leu310GluVal converts a nonpolar
residue (Leu) into two residues, one negatively charged (Glu) and the
other nonpolar (Val).
Two previously reported RDH5 mutations, Ser73Phe and
Gly238Trp, were found to have 90% lower enzymatic activity than the
wild-type enzyme in transfected COS-1 cells.7
Although we
did not perform a similar analysis of the mutations, it is likely that
they also result in reduced enzyme activity. 11-cis Retinol
dehydrogenase has two potential membrane-anchoring
domains.11
The position of a highly conserved
cofactor-binding motif is located at residues 35 to 41, and a presumed
catalytic domain is located at residues 175 to 179.11
Although residue Val264 is not within these domains, it is highly
conserved in the superfamily of short-chain reductase
dehydrogenases.13
The second mutation we found affects
residue Leu310, which is located in a potential membrane-anchoring
domain.11
Because this change alters two residues from
nonpolar to negative charged and polar, this mutant protein may not be
able to anchor to the membrane. Therefore, because the intracellular
localization or membrane topology of this protein may be altered, the
enzyme activity of the protein may decrease. However, because we did
not perform biochemical analysis of these mutant proteins, further
studies are needed to determine how the mutations affect the function
of RDH5.
All the patients described in this study had similar clinical findings:
small white deposits at the level of the RPE, normal visual acuity,
full visual fields, and very slow dark adaptation. Because the patients
were aged 45 and 55 years, it is unlikely that their disease was
progressive. These clinical findings are similar to those previously
reported in which mutations in RDH5 were
found.7
8
Although the mutations found in RDH5
cause fundus albipunctatus in both the present and previous
reports,7
8
recently a white family with fundus
albipunctatus was reported to show no mutations in
RDH5.14
Therefore, fundus albipunctatus may be
genetically heterogeneous.
|
Acknowledgements
|
|---|
The authors thank Kaorn Nakano and Sachiko Takiuchi for
technical assistance and Thaddeus P. Dryja and Terri L. McGee for
helpful discussions.
|
Footnotes
|
|---|
3 EH and YI contributed equally to this work. 
Supported by Grant-in-Aid 10671643 for Scientific Research from the Ministry of Education, Culture, and Science of Japan (SY) and a grant for research on eye and ear science, immunology, allergy, and organ transplantation from the Ministry of Health and Welfare (SY, YT), Tokyo, Japan.
Submitted for publication February 16, 2000; revised April 19, May 30, and July 25, 2000; accepted July 28, 2000.
Commercial relationships policy: N.
Corresponding author: Hiroyuki Morimura, Department of Ophthalmology Osaka University Medical School, Room E7, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. morimura{at}ophthal.med.osaka-u.ac.jp
|
References
|
|---|
-
Lauber, H. (1910) Die sogenannte Retinitis punctata albescens Klin Monatsbl Augenheilkd 48,133-148
-
Souied, E, Soubraner, G, Benlian, P, et al (1996) Retinitis punctata albescens associated with the Arg135Trp mutation in the rhodopsin gene Am J Ophthalmol 121,19-25[Medline][Order article via Infotrieve]
-
Kajiwara, K, Sandberg, MA, Berson, EL, Dryja, TP (1993) A null mutation in the human peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens Nat Genet 3,208-212[Medline][Order article via Infotrieve]
-
Weleber, RG, Carr, RE, Murphey, WH, Sheffield, VC, Stone, EM (1993) Phenotypic variation including retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in a single family with a deletion of codon 153 or 154 of the peripherin/RDS gene Arch Ophthalmol 111,1531-1542[Abstract]
-
Burstedt, MS, Sandgren, O, Holmgren, G, Forsman–Semb, K. (1999) Bothnia dystrophy caused by mutations in the cellular retinaldehyde-binding protein gene (RLBP1) on chromosome 15q26 Invest Ophthalmol Vis Sci 40,995-1000[Abstract/Free Full Text]
-
Morimura, H, Berson, EL, Dryja, TP (1999) Recessive mutations in the RLBP1 gene encoding cellular retinaldehyde-binding protein in a form of retinitis punctata albescens Invest Ophthalmol Vis Sci 40,1000-1004[Abstract/Free Full Text]
-
Yamamoto, H, Simon, A, Eriksson, U, Harris, E, Berson, EL, Dryja, TP (1999) Mutations in the gene encoding 11-cis retinol dehydrogenase cause delayed dark adaptation and fundus albipunctatus Nat Genet 22,188-191[Medline][Order article via Infotrieve]
-
Gonzalez–Fernandez, F, Kurz, D, Bao, Y, et al (1999) 11-cis Retinol dehydrogenase mutations as a major cause of the congenital night-blindness disorder known as fundus albipunctatus Mol Vis 5,41-46[Medline][Order article via Infotrieve]
-
Schoenlein, RW, Peteanu, LA, Mathies, RA, Shank, CV (1991) The first step in vision: femtosecond isomerization of rhodopsin Science 254,412-415[Abstract/Free Full Text]
-
Dowling, JE (1960) Chemistry of visual adaptation in the rat Nature 188,114-118[Medline][Order article via Infotrieve]
-
Simon, A, Romert, A, Gustafson, AL, McCaffery, JM, Eriksson, U. (1999) Intracellular localization and membrane topology of 11-cis retinol dehydrogenase in the retinal pigment epithelium suggest a compartmentalized synthesis of 11-cis retinaldehyde J Cell Sci 112,549-558[Abstract]
-
Simon, A, Lagercrantz, J, Bajalica–Lagercrantz, S, Eriksson, U. (1996) Primary structure of human 11-cis retinol dehydrogenase and organization and chromosomal localization of the corresponding gene Genomics 36,424-430[Medline][Order article via Infotrieve]
-
Jornvall, H, Persson, B, Krook, M, et al (1995) Short-chain dehydrogenases/reductases (SDR) Biochemistry 34,6003-6013[Medline][Order article via Infotrieve]
-
Yamamoto, H, Fishman, GA, Berson, EL, Dryja, TP. (2000) Three novel mutations in the RDH5 encoding 11-cis retinol dehydrogenase in patients with fundus albipunctatus [ARVO Abstract] Invest Ophthalmol Vis Sci 41(4),S615Abstract nr 3265
This article has been cited by other articles:
|
|
|
|
 
Y. Niwa, M. Kondo, S. Ueno, M. Nakamura, H. Terasaki, and Y. Miyake
Cone and Rod Dysfunction in Fundus Albipunctatus with RDH5 Mutation: An Electrophysiological Study
Invest. Ophthalmol. Vis. Sci.,
April 1, 2005;
46(4):
1480 - 1485.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Nakamura, J. Lin, and Y. Miyake
Young Monozygotic Twin Sisters With Fundus Albipunctatus and Cone Dystrophy
Arch Ophthalmol,
August 1, 2004;
122(8):
1203 - 1207.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Sekiya, M. Nakazawa, H. Ohguro, T. Usui, N. Tanimoto, and H. Abe
Long-term Fundus Changes Due to Fundus Albipunctatus Associated With Mutations in the RDH5 Gene
Arch Ophthalmol,
July 1, 2003;
121(7):
1057 - 1059.
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. I. Brueggemann and J. M. Sullivan
HEK293S Cells Have Functional Retinoid Processing Machinery
J. Gen. Physiol.,
May 28, 2002;
119(6):
593 - 612.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G.-F. Jang, J. P. Van Hooser, V. Kuksa, J. K. McBee, Y.-G. He, J. J. M. Janssen, C. A. G. G. Driessen, and K. Palczewski
Characterization of a Dehydrogenase Activity Responsible for Oxidation of 11-cis-Retinol in the Retinal Pigment Epithelium of Mice with a Disrupted RDH5 Gene. A MODEL FOR THE HUMAN HEREDITARY DISEASE FUNDUS ALBIPUNCTATUS
J. Biol. Chem.,
August 24, 2001;
276(35):
32456 - 32465.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Liden, A. Romert, K. Tryggvason, B. Persson, and U. Eriksson
Biochemical Defects in 11-cis-Retinol Dehydrogenase Mutants Associated with Fundus Albipunctatus
J. Biol. Chem.,
December 21, 2001;
276(52):
49251 - 49257.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
