Download Phenotypes of stop codon and splice site rhodopsin

Phenotypes of Stop Codon and Splice Site Rhodopsin
Mutations Causing Retinitis Pigmentosa
Samuel G. Jacobson,* Colin M. Kemp,* Artur V. Cideciyan,* Jennifer P. Macke,\
Ching-Hwa Sung\, and Jeremy Nathans^
Purpose. To understand the pathophysiology of retinitis pigmentosa caused by mutations in
the rhodopsin gene that lead to truncation of the protein.
Methods. Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the
glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at
least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels.
Results. There was a range of severity of disease expression in each family, some heterozygotes
having moderate or severe retinal degeneration and others with a mild phenotype. The mildly
affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and
reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course
of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter
mutations both had prolonged recovery of sensitivity, but the time course was different in the
two genotypes.
Conclusions. There is allele specificity for the pattern of retinal dysfunction in the Q64ter,
intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three
mutations suggests the mutant opsins interfere with normal rod cell function, and there is
subsequent rod and cone cell death. Invest Ophthalmol Vis Sci. 1994;35:2521-2534.
XVetinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerations, some of which
are caused by mutations in the gene encoding rhodopsin.1 Most of the rhodopsin gene mutations responsible for RP are point mutations or small deletions, and
all but one cause autosomal dominant RP (adRP) (for
example, refs. 2-6). The exception is a stop codon
mutation, glutamic acid-249-to-ter (E249ter), recently
reported as a putative null allele that causes autosomal
From the *Department of Ophthalmology, University of Miami School of Medicine,
Batcom Palmer Eye Institute, Miami, Florida, and the ^Departments of Molecular
Biology and (ienetics, mid Neuroscience, Howard Hughes Medical Institute, Johns
Hopkins University School of Medicine, Baltimore, Maryland.
Supported in part by Public Health Service research grant EY05627 (SGJ); the
National Retinitis Pigmentosa Foundation, Inc., Baltimore, Maryland; The Chatlos
Foundation, Inc., Longwood, Florida; and the Howard Hughes Medical Institute,
Belhesda, Maryland. Dr. Jacobson is a Research to Prevent Blindness Dolly Green
Scholar.
Submitted for publication October I, 1993; revised November 5, 1993; accepted
November 12, 1993.
Proprietary interest category: N.
Reprint requests: Dr. Samuel G. Jacobson, Bascom Palmer Eye Institute, 1638
N.W. 10th Avenue, Miami, FL 33136.
liivcsiigativc Ophthalmology & Visual Science, April 1994. Vol. 35, No. 5
Copyright-© Association for Research in Vision and Ophthalmology
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recessive RP (arRP). Heterozygotes with the E249ter
rhodopsin mutation had normal clinical examinations
but mild rod photoreceptor-mediated functional disturbances.7 This finding of null alleles carried in single
dose in apparently unaffected individuals7 prompted
the hypothesis that the rod photoreceptor can remain
healthy with only half the normal level of wild-type
rhodopsin, whereas it cannot in the presence of abnormal rhodopsin due, for example, to a missense mutation.8
A recent search for rhodopsin mutations in 282
patients with RP6 revealed two families, one with patients heterozygous for a stop codon mutation, glutamine-64-to-ter (Q64ter), and another with patients
heterozygous for the intron 4 splice site mutation
guanosine43!<;>-to-thymidine, a mutation described previously in a possible carrier of arRP.7 These potential
null alleles, however, were found in families with RP
that had at least three generations of affected
members,6 indicating that these alleles are not innocu-
2521
2522
Investigative Ophthalmology 8c Visual Science, April 1994, Vol. 35, No. 5
ous in single dose and that these heterozygotes have
adRP.
To understand more about the pathophysiology
of rhodopsin gene mutations in RP that could lead to
truncation of the protein, we studied the functional
phenotypes of patients with the Q64ter mutation and
the intron 4 splice site mutation. We also compared
the patterns of disease expression in these mutations
with the pattern in heterozygotes with a mild phenotype who carry another rhodopsin stop codon mutation, glutamine-344-to-ter (Q344ter), reported previously. 2910
ocular examination, and most patients had Goldmann
kinetic perimetry, dark- and light-adapted static
threshold perimetry, and full-field ERGs using a clinical protocol. The patients with relatively mild disease
also had dark adaptometry, measurements of rod-isolated ERG a- and b-waves, and fundus reflectometry.
Informed consent was obtained from the patients and
from normal subjects involved in the study after the
nature of the procedures had been explained fully.
The research procedures were in accordance with institutional guidelines and with the Declaration of Helsinki.
Visual Function Tests
MATERIALS A N D METHODS
Static threshold perimetry in the dark- and lightadapted states was performed using techniques previously described. 1112 For dark-adapted perimetry, 75
loci (12° grid) in the visual field were tested with 650
nm and 500 nm stimuli (target size V). Photoreceptor
mediation at each locus was determined from the sensitivity differences between the two stimulus colors, and
rod sensitivity losses were calculated based on 500 nm
test results in comparison to normal mean values. For
light-adapted (10 cd-rrT 2 white background) perime-
Subjects
The 19 patients in this study were from three families.
Thirteen of 14 patients from the family with the
Q64ter mutation (all but patient 3, Table 1), the three
patients from the family with the intron 4 splice site
mutation, and both patients with the Q344ter mutation had previously participated in molecular genetics
investigations that determined that they were heterozygotes with these mutations. 2 6 All patients underwent
TABLE
l. Clinical Characteristics of the Patients
Visual Acuity*
Patient
No.
Q64ter
1
2
3
4
5
6
7§
7"
8
9
10
11
12
13
14
Intron 4 splice site
15
16
17
Generation
No.
Age (yr)/
Sex
RE
LE
IV
IV
III
III
II
III
II
II
II
11
11
II
11
I
I
1 1/M
J3/F
24/M
32/F
34/M
35/F
30/F
37/F
43/F
64/M
65/F
71/M
73/F
75/F
76/M
20/25
20/40
20/20
20/40
20/30
20/30
20/20
20/50
20/40
20/30
2/200
20/60
20/60
LP
7/200U
20/30
20/30
20/20
20/40
20/30
20/30
20/20
20/100
20/30
20/50
LP
20/100
20/60
LP
2/200
III
II
I
21/F
46/M
76/F
20/20
20/50**
HM
20/20
20/20
NLPH
Kinetic Visual
Field ExtentfX
(V-4e/I-4e)
Fundus
Appearance^
41/35
37/5
98/28
42/21
92/72
15/2
94/75
91/43
86/<I
55/<l
PR
PR
PR
PR
N
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
U/U
<I/U
<l/<]
u
31/U#
94/94
95/60
U
N
N
PR
N = no abnormalities; PR = pigmentary retinopathy; HM = hand motions vision; NLP = no light perception; LP = light perception; U =
immeasurable.
* Best corrected visual acuity.
t Similar in the two eyes, unless specified.
X Expressed as a percent of normal mean; 2 SD below normal equals 90% for V-4e and 88% for I-4e.19
§ Visit in 1985.
11
Visit in 1992.
11 Glaucoma.
#
Nonglaucomatous eye.
** Strabismic amblyopia.
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Stop Codon Rhodopsin Mutations and RP
try, the same 75 loci were tested with a 600 nm target.
Cone sensitivity losses at each locus were determined
by comparison with normal mean values.
Dark adaptometry was tested with 500 nm and
650 nm stimuli (target size V) at 12° in the inferior
visual field. Baseline dark-adapted thresholds were determined after at least 3 hours of dark adaptation on a
day before exposure to any bright lights. A yellow
bleaching light (wavelengths > 520 nm) was delivered
with Maxwellian optics using a fundus camera (Carl
Zeiss, Wetzlar, Germany); the 30° diameter field was
centered on the test locus. For each patient, the recovery of sensitivity was measured after retinal exposures
of 7.8, 6.9, and 6.3 log scot-td • s. These exposures are
expected to bleach about 99, 50 and 15% of the rhodopsin originally present, respectively. Further details
of the method have been reported.13
The time courses of dark adaptation in the patients were analyzed using a model shown to provide
an accurate description of the kinetics of recovery of
sensitivity in normals after adapting lights that bleach
from as little as 1% to greater than 99% of the rhodopsin originally present.1415 Lamb's scheme postulates
that the control of rod sensitivity results from the persistent presence of small amounts of R* (the activated
form of photolyzed rhodopsin) after extinction of the
adapting light. Lamb proposed that the R* is produced from relatively long-lived rhodopsin photoproducts Sj, via reverse reactions. The model thus consists
of three sequential first-order reactions, each of which
is weakly reversible and one of which saturates (i.e.,
becomes zero-order) after intense light adaptation:
Rhodopsin
light
k 21
S 2 ^==± S
k 32
where interconversion of S 2 a n d S 3 is rate limited, with
a half-saturating value for S 2 of S 2sal . T h e model does
not identify the individual p h o t o p r o d u c t s , which in
principle could include one or more forms of phosphorylated opsin and/or opsin to which arrestin is
bound. Though it does not have a comprehensive
foundation of specified molecular reactions involving
the rhodopsin molecule, it provides a relatively simple
basis for describing quantitatively the kinetic abnormalities observed in several forms of RP, with an accuracy and level of detail that is unattainable using conventional mathematical schemes proposed for rod
dark adaptation in the intact eye (for example, refs. 16
and 17). In particular, it enables various time domains
(each associated with the relative abundance of one of
the species Sls S2, or S3) during dark adaptation to be
identified. As a result, the extent to which each of
these is affected in patients with rhodopsin mutations
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2523
can be individually assessed. Solutions of the set of
first-order differential equations describing the model
were obtained by numerical integration using the
Runga-Kutta method,18 and for each subject values
for the parameters were obtained by minimizing the
errors of the fits to the rod recovery data for all three
bleaches.
Full-field ERGs were performed using bipolar
Burian-Allen contact lens electrodes and a computerbased system previously described.19'20 Suprathreshold stimuli were used to elicit a rod ERG (blue flash of
—0.1 log scot-td *s, dark-adapted); a mixed cone and
rod ERG (white flash of 5.4 cd • s • m~2, dark-adapted);
and a cone flicker ERG (29 Hz white light flashes of
0.64 cd'S«m~2, on a white background of 6.9
cd • m~2). ERGs were also elicited in the dark-adapted
state to different intensities of blue (Wratten 47B; Kodak, Rochester, NY) light flashes over a 3 log unit
range (up to 1.8 log scot-td • s). Waveforms were measured conventionally as follows: b-wave amplitude
from baseline or the a-wave trough (when present) to
the major positive peak; implicit time from stimulus
onset to the major peak of the response; and for the
cone flicker ERG, amplitude from negative to positive
peak and timing to the positive peak. The Naka-Rushton equation [V = Vmax*In/(In + Kn)] was fitted to the
measured b-wave amplitudes from the intensity series
to blue light flashes. In the equation, V is rod b-wave
amplitude; Vmax, the amplitude at response saturation;
I, the stimulus intensity; K, the intensity at half Vm;ix;
and n, the exponent responsible for the slope of the
function.
Rod-isolated ERGs to high-intensity stimuli were
performed using unipolar Burian-Allen contact lens
electrodes and recording and analysis methods previously published.21 In brief, pairs of scotopically
matched waveforms to blue (Wratten 47A) and red
(Wratten 26) flashes were digitally subtracted to give a
cone ERG, that was then subtracted from the response
to a photopically matched blue flash (double subtraction technique22). Responses to a range of intensities
from 2 log scot-td • s to 4.5 log scot-td • s in 0.3 log unit
steps were recorded.
The photoreceptor generated component of the
rod-isolated ERG, PHI23 or P324, was estimated by fitting a mathematical model to the leading edge of the
a-waves in the intensity series. The model consisted of
a family of delayed Gaussian functions of time and
stimulus intensity25"28:
t) = Rr
-exp[---I-<7-(t-td)2
0)
where Rniax is the maximum response amplitude in nV;
I, the energy of a brief flash in scot-td • s; a, the sensitivity in scot-td"1 • s~3; t, the time after flash onset in sec-
Investigative Ophthalmology 8c Visual Science, April 1994, Vol. 35, No. 5
2524
onds; and td, a brief time delay in seconds that approximates the initial stages of the transduction cascade as
well as delays due to the recording apparatus. The sensitivity parameter a is equal to the product of k^, which
is the number of isomerizations produced per rod per
scot-td • s of retinal illuminance, and A, which is the
amplification constant in s"2. The amplification constant A is the product of the rate of activated phosphodiesterase production per isomerized rhodopsin molecule, the rate of cGMP hydrolysis per activated phosphodiesterase, and the Hill coefficient governing the
fraction of open channels.25"27 The value of k^, is estimated to be approximately 5 in normal subjects.25
Parameters of the P3 model (Rmax, o, and td) were
determined in two steps. First, the waveforms were
edited to make the pre-stimulus baselines coincide and
were cut at the time when the b-wave intrudes. Next,
the edited waveforms were used to find automatically
(Matlab 4.0, The Math Works, Natick, MA) the two
parameter values (a and td) that minimize the squared
error between the model and the ensemble of waveforms. The parameter Rmax was set equal to the largest
negative amplitude in the series. To permit independent comparisons of Rnuix and a in patients and normal
subjects, the response amplitude predicted by the P3
model was plotted against stimulus intensity for a fixed
time.2'' For afixedtime after the time delay (t — td = T),
the photoreceptor response shown in equation 1
reaches half-maximum response at the intensity
2•ln(2)
50%
(2)
On a graph of response amplitude versus log stimulus
energy, a change in Rmax would correspond to a vertical scaling and a change in a would correspond to a
horizontal shift.
Imaging fundus reflectometry was performed
with instrumentation and methods already described.30" Rhodopsin losses in the patients were determined by comparison of their double difference values (at 520 nm) with those from normal subjects at
matched retinal locations. To study the relationship
between the rhodopsin levels and rod-mediated sensitivity, dark-adapted static perimetric measurements
with the 500 nm stimulus were made at 25 loci within
the retinal region tested with fundus reflectome t r y
10..3
RESULTS
The schematic drawing of the rhodopsin molecule in
Figure 1 shows the sites of the Q64ter, intron 4 splice
site, and Q344ter mutations. The Q64ter mutation
would encode a truncated protein missing six of the
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Q344ter
Q64ter
G-T,bp4335
l. Schematic drawing of the rhodopsin molecule.
Amino acids are shown as circles. The sites of the mutations
carried by the patients in this study are indicated.
FIGURE
seven transmembrane domains, including the site of
attachment of 11-cis retinal.6 The substitution in the
donor splice site of intron 4 could lead to an abnormal
carboxy terminal region of the molecule. The Q344ter
mutation encodes a protein missing the last five amino
acids.20
Table 1 shows some clinical characteristics of the
patients in this study. In the family with the Q64ter
mutation, 12 of the 14 heterozygotes examined have
ophthalmoscopic features of RP with attenuated retinal vessels, pigmentary retinopathy, and a waxy pale
appearance to the optic nerve head. P5 has a normal
ophthalmoscopic examination, and his sister, P7, has
cystoid macular edema and only a few pigmentary
changes in the peripheral retina. Visual acuities and
kinetic visual fields range from normal or nearly normal to moderately or severely abnormal.
In the family with the intron 4 splice site mutation,
PI5 and PI6 have normal-appearing fundi, but PI7
has ophthalmoscopic evidence of an advanced stage of
RP in both eyes. Visual acuities ranged from normal
(PI5 in both eyes; PI6 in his non-amblyopic eye) to
severely abnormal (PI 7 in her eye without glaucoma).
Kinetic fields were normal in PI5, slightly subnormal
in PI6, and reduced to a small central island in PI7.
Records obtained from previous examinations of PI 7
indicated that Goldmann kinetic perimetry (V-4e target) 15 years earlier showed a central island and a temporal peripheral island separated by a nearly complete
annular midperipheral scotoma; another field 5 years
earlier showed only a small central island of vision.
In our previous description of the phenotype of a
family with RP caused by the Q344ter mutation, we
noted that three siblings carrying the mutation had
normal ocular examinations, normal visual acuities
and kinetic fields, and abnormal rod function but normal cone function (patients 1 to 3 in ref. 10). In the
present study, two of the patients, designated as PI8
2525
Stop Codon Rhodopsin Mutations and RP
and PI9 (representing patients 2 and 3, respectively,
in ref. 10), were reexamined with further visual function tests to permit comparison with results obtained
from the patients with mild phenotype from the families with Q64ter and splice site mutations.
Figure 2 shows results of kinetic and static perimetry in three family members with the Q64ter mutation,
representing different degrees of disease expression.
P5 has a normal extent of visual field with kinetic perimetry using the V-4e target but a slightly reduced
extent with the I-4e target (Table 1). There is rod sensitivity loss (mean loss, 11.3 dB; SD 2.2 dB) across most
of the visual field. P4 has a kinetic visual field with
reduced extent in the periphery with both target sizes
(Table 1); rod sensitivity losses are far greater (mean of
the 46 loci with measurable function, 32.9 dB; SD 9.2
dB) than in P5. The kinetic field of P2, the daughter of
P4, using the V-4e target has a central island separated
from an island in the temporal peripheral field by an
incomplete annular midperipheral scotoma; with the
I-4e target, the field is limited to only a central island.
Rod sensitivity is measurable only centrally and in the
temporal periphery and is reduced by between 2 and 3
log units at these loci (mean of the 22 loci with measurable function, 30 dB; SD 11.2 dB). Mean cone sensitivity losses across the visual field for P5 were 0 dB (SD
2.1 dB); mean of loci with measurable function for P4
were 9.1 dB (SD 3.8 dB; n = 30 loci) and for P2 were
6.0 dB (SD 5.9 dB; n = 15 loci).
Figure 3 shows perimetric results in P7, the sister
of P5, on two visits separated by about 7 years. In
1985, the patient had a normal extent of kinetic field
with the V-4e target but a slightly reduced extent with
the I-4e target (Table 1). Rod sensitivity loss at this
time was 10.0 dB (SD 4.8 dB). In 1992, the kinetic field
to the I-4e is more reduced in extent (Table 1), and rod
sensitivity loss had increased (mean 17.6 dB; SD 5.8
dB). To determine if this progression of rod sensitivity
Q64ter
PATIENT 5
PATIENT 4
PATIENT 2
KINETIC PERIMETRY
ROD SENSITIVITY LOSS
0)
2,
3624-
12 0 -
£
zJ
U
o
o
12-
24 3648-
UJ
N 48 36 24 12 0 12 24 36 48 60 72 T N 48 36 24 12 0 12 24 36 48 60 72 T N 48 36 24 12 0 12 24 36 48 60 72 T
ECCENTRICITY [deg]
FIGURE 2. Kinetic perimetry (upper) and dark-adapted static threshold perimetry (lower) in
three patients with the Q64ter mutation. V (target area 64 mm2) and I (target area 0.25 mm2)
targets at intensity 4-e (318 cd • m~2) were used for kinetic perimetry. Results of static perimetry are displayed as gray scales of rod sensitivity losses. Gray scales have 16 levels representing
0 to 35 dB (1 dB equals 0.1 log units) sensitivity loss. White is 0 to 2 dB loss, and black is
greater than 35 dB loss. Physiological blind spot is shown as a black square at 12° in the
temporal field.
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Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
2526
Q64ter
PATIENT 7,1985
PATIENT 7,1992
KINETIC PERIMETRY
ROD SENSITIVITY LOSS
0)
36-
3^
>
o
24-
E
z
UJ
12 -
o
o
12-
0 -
2436-
48 -
UJ
T 72 60 48 36 24 12
0 12 24 36 48 N T 72 60 48 36 24 12
0 12 24 36 48 N
ECCENTRICITY [deg]
FIGURE 3. Kinetic perinietry and dark-adapted static threshold perimetry in P7, a patient with
the Q64ter mutation, on two visits separated by about 7 years. The data are displayed as in
Figure 2.
loss affected some regions of the visual field more than
others, we divided the field into three regions and calculated the average of rod sensitivity losses within
these regions. At eccentricities <30°, there was about
5 dB loss between visits; between 30° and 60°, there
was nearly 10 dB loss; and at eccentricities >60°,
about 7 dB loss occurred. This suggests that in the
7-year interval between visits, the midperipheral field
had more sensitivity loss than peripheral and central
fields. Data for cone sensitivity across the visual field
was available for only the later visit; mean cone sensitivity loss was 3.7 dB (SD 4.1 dB).
Figure 4 shows results of kinetic and static perimetry in two mildly affected patients representing two
generations with the intron 4 splice site mutation
(PI5, PI6) and a patient with the Q344ter mutation
(PI9). PI5 and PI9 both have normal kinetic fields,
whereas PI6 shows a slightly reduced extent with the
I-4e target (Table 1). All three patients show some rod
sensitivity losses across the visual field. Mean rod sensitivity losses were as follows: PI5, 7.4 dB (SD 2.0 dB);
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P16, 7.8 dB (SD 2.7 dB); and P19, 9.9 dB (SD 3.1 dB).
Cone sensitivity losses were as follows: PI 5, 0.5 dB (SD
1.6 dB); P16, 2.7 dB (SD 2.0 dB); and P19, 0.2 dB (SD
1.7 dB). The two other siblings of PI 9 showed similar
results with dark- and light-adapted static perimetry to
their sister.10
Figure 5 shows dark adaptometry results in two
representative normal subjects and six patients, two
from each genotype. For the light-adapting exposure
used, which is expected to bleach ~99% of the visual
pigments originally present within the test area,13 the
normal subjects recovered completely to their baseline
dark-adapted sensitivity levels after about 55 minutes
in darkness. P5 and P7, who carry the Q64ter mutation, showed a similar time course, with no delay of
either the appearance of the rod recovery branch or of
the attainment of their baseline sensitivities (which are
reduced by 7 dB and 9 dB, respectively). PI 5 and PI6,
who carry the splice site mutation, both had slower
recovery than in normal subjects, requiring about 90
minutes to return to within 1 dB of their pre-bleach
2527
Stop Codon Rhodopsin Mutations and RP
SPLICE SITE
PATIENT 15
Q344ter
PATIENT 16
PATIENT 19
KINETIC PERIMETRY
ROD SENSITIVITY LOSS
s
36 -
3^
>H
24 12 -
O
0 -
c
12 -
hZ
UJ
o
o
r1 .•
24 36-
48 -
UJ
N 48 36 24 12 0 12 24 36 48 60 72 T T 72 60 48 36 24 12 0 12 24 36 48 N T 72 60 48 36 24 12 0 12 24 36 48 N
ECCENTRICITY [deg]
FIGURE 4. Kinetic perimetry and dark-adapted static threshold perimetry in two patients who
carry the intron 4 splice site mutation (PI 5, PI6) and a patient with the Q344ter mutation
(PI 9). The data are displayed as in Figures 2 and 3.
sensitivity levels. Recovery of rod sensitivity in the two
patients with the Q344ter mutation is also prolonged
and, in each case, takes more than 2 hours to return to
within 1 dB of the pre-bleach level.
The curves fitted to the time course of recovery of
rod function in the patients and the normal subjects
are derived from the scheme proposed by Lamb.14'32
The kinetic parameters used to fit each set of data are
shown in Table 2. When there were only small differences between the kinetics of the recovery curves for
patients with the same mutation, a single set of parameters was used to generate the curve that describes
them. In the case of the patients with the splice site
mutation, curves were fitted to the data of PI5 and
PI6 individually; with one exception (k2i), all abnormalities were similar in the two patients. Although
some of the parameters in the data from the patients
with the Q344ter mutation are similar to those in the
data from patients with the splice site mutation, there
are substantial quantitative differences between the
sets associated with the two genotypes.
Electroretinography using a clinical protocol
showed that rod ERGs, mixed cone and rod ERGs, and
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cone flicker ERGs were abnormal to varying degrees
in the seven members of the family with the Q64ter
mutation who were tested. Patients 4, 6, 8, 12, and 14
had no detectable responses to any of the stimuli. P5
and P7 had rod b-waves with reduced amplitude and
normal implicit times. Cone flicker amplitude was normal, but timing was delayed in these patients. Serial
data on P7 showed further reduction in rod b-wave
amplitude and greater prolongation of cone flicker
timing between visits, separated by 7 years. In the family with the splice site mutation, ERGs in PI5 and PI6
showed reduced rod b-wave amplitudes and normal or
slightly delayed timing; cone ERGs were normal.
ERGs in PI7 were not detectable on an examination
15 years earlier. Results with these stimuli in the patients with the Q344ter mutation have been published.10 They showed mainly rod amplitude abnormalities.
Figure 6A shows the first 15 ms of the rod-isolated
responses in a normal subject and in three patients,
each representing a different genotype. These patients
had the mildest disease expression among those examined in their family. The P3 model has been fitted to
2528
Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
10
I
NORMAL
Q64ter
20
30
40
CD
°°S5o o
"S&S»n) oo
K n
Q
>1
OUil)lM>0«>-00
0
*
,
SPLICE SITE
I
v,
Q344ter
U)
Z 20
LJJ
CO
30
40
v
<•*•••••••»
50
60 PB 0
20
40
60
1
2h
PB 0
20
40
TIME
mm
3h
60
1
FIGURE 5. Dark adaptometry results after bleaching of 99% rhodopsin at 12° in the inferior
field in two normal subjects (upper left); P5 (unfilled squares) and P7 (filled squares) with the
Q64ter mutation (upper right); PI5 (unfilled circles) and PI6 (filled circles) with the splice
site mutation (lower left); and PI8 (unfilled triangles) and PI9 (filled triangles) with the
Q344ter mutation (lower right). Each panel also includes curves illustrating thefitof a model
for kinetic analysis of rod dark adaptation to the data from the normal subjects and each
genotype, using the parameters given in Table 2. PB, pre-bleach or baseline dark-adapted
sensitivity level. Note the compressed time-scale used for times greater than 1 hour after the
bleach.
the responses from an intensity series. P5, representing the family with Q64ter mutation, and PI8, representing the family with the Q344ter mutation, have
lower maximum amplitude than the normal subject,
whereas PI5, with the splice site mutation, has a response with amplitude closer to that of the normal.
TABLE 2.
Parameters* Describing the Kinetics of Rod Dark Adaptometry
Patient
No.
Q64ter
5
7
Intron 4 splice site
15
16
/~\O A A *. —„
^;o44ter
18
19
Normal
Table 3 lists the P3 model parameters (Rmax, a, td) for
six patients, two from each family, and, for comparison, the mean and standard deviation for a group of
normal subjects. Rmax offiveof the six patients (except
PI5) fell outside the range of the normal subjects; a
and td were within the normal range for all six patients.
*»t
"•23
k32
k34
l3
2sat+
0.05
0.05
0.00025
0.0006
0.0125
0.012
0.00035
0.0004
0.00275
0.00275
0.14
0.14
0.04
0.04
0.0022
0.004
0.009
0.0085
0.000008
0.000009
0.0011
0.0011
0.42
0.42
0.04
0.04
0.045
0.00055
0.00085
0.00075
0.006
0.006
0.0125
0.00005
0.00003
0.00005
0.00075
0.00075
0.00275
0.33
0.33
0.13
* Parameters were obtained using the model proposed by Lamb 14 , k10 was treated as invariant from normal in all cases.
f ky values are in units of s"1.
X S25at is the half saturating concentration of S2-
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
2529
Stop Codon Rhodopsin Mutations and RP
NORMAL
^
-300
LLJ
Q -400
J
Q.
SPLICE SITE
0
2
** -100
15
0
5
TIME [ms]
B
i 500
t; 400
<
§ 200
I 100
""
2.5
3
3L5
4
STIMULUS ENERGY [log scot Id s]
4.5
FIGURE 6. (A) Rod-isolated a-waves to different stimulus intensities in a normal subject and patients with the Q64ter
(P5), the splice site (PI5), and the Q344ter (PI8) rhodopsin
mutations. The smooth curves are a family of delayed Gaussian functions fitted to the leading edges of a-waves in the
intensity series. The stimulus energies were O = 4.5, • =
4.2, V = 3.9, T = 3.6, • - 3.3, • = 3.0, A - 2.7, • = 2.4, 0
= 2.1 log scot-td • s. (B) Graph of P3 model amplitude at the
fixed time of 5 ms after time delay versus log stimulus energy. Solid lines represent the six patients whose data are in
Table 3, and the dashed line represents the mean normal.
Vertical lines correspond to the intensity producing half
maximum response, I50%. Error bar on vertical axis is the
mean normal Rmax — 2 SD; bar on horizontal axis is mean
normal I50% ± 2 SD.
The relationship between Rniax and a is shown in Figure 6B, which plots the P3 model amplitude at a fixed
time versus the log stimulus energy for six patients,
two from each genotype. The vertical lines denote the
I5()% values for the patients.
In Figure 7 are graphs of rod ERG b-wave amplitudes at different intensities of blue light flashes in the
dark-adapted state in six patients, two representatives
of each genotype, compared to normal subjects. Both
patients with the Q64ter mutation show a reduced
VnKlx and an abnormal K. The serial data in P7 are
notable in that they provide some information about
the natural history of rod ERG change in the Q64ter
Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017
mutation; disease progression in this mutation appears to lead to more reduction of Vniax and a further
shift in K. The patients with the splice site mutation
had abnormal Vniax and K. One of the two patients with
the Q344ter mutation also followed this pattern,
whereas another fell just within the normal limits (outside the ± 1 SD range but inside the ± 2 SD range) for
both Vmax and K. The parameters derived from the
fitting of the Naka-Rushton equation to the rod ERG
intensity series are given in Table 3. It is of interest
that the ratio of b-wave Vmax to a-wave Rniax is about 1.0
or greater for patients with Q64ter and Q344ter mutations and for normal subjects. In the splice site mutation patients, however, the ratio of these parameters
is about 0.5, and there were negative waveforms to all
high-intensity stimuli. This suggests there is dysfunction not only at the rod outer segment but also at or
proximal to the photoreceptor terminal region, such
as has been recently demonstrated in patients with RP
of unknown genotype.21
Imaging fundus reflectometry was performed on
P5 and P7 from the family with the Q64ter mutation
and on PI 5 and PI6 from the family with the splice site
mutation. Figure 8 shows the relation between rhodopsin levels and psychophysically measured rod sensitivity losses in the four patients. In P5 and P7, measured pigment densities were considerably reduced
from normal by a relatively constant amount. In PI5
and PI6, there was greater variation of densities
within the measurement area. For all patients, the data
points lie close to the line illustrating the predicted
relationship for rod sensitivity losses caused by decreased light absorption as rhodopsin levels diminish.
A similar pattern of results was found in patients with
the Q344ter mutation.10
DISCUSSION
The ocular examination results in the patients heterozygous for the Q64ter, intron 4 splice site, and
Q344ter mutations in the rhodopsin gene showed that
there was a range of severity of disease expression in
each genotype. Some heterozygotes from each family
had pigmentary retinopathy with reduced visual acuity
and diminished visual field extent, whereas others had
a normal ophthalmoscopic appearance with normal
acuity and full kinetic fields. Rod-specific visual function test results indicated that even the patients who
were apparently unaffected clinically had abnormal
rod-mediated function.
Dark-adapted perimetry has shown specific patterns of rod sensitivity loss, such as diffuse or altitudinal patterns, in previous studies of adRP patients
with rhodopsin mutations.10'13 Regional retinal differences in disease severity were not discernible from the
available data in the families with splice site and
Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
2530
TABLE
3. Rod Isolated ERG Results
A-wave*
Patient
Rmax
0"
No.
(*V)
(scot-td~' -s~3)
168
NA
99
32.2
B-wave-f
Vmax
h
(msec)
logK
(log scot-td -s)
n
Q64ter
5
n7§
18.8
3.3
NA
3.0
166
195
133
0.17
-0.29
0.17
0.84
0.96
0.75
373
313
43.5
33.5
3.4
3.5
176
151
0.05
-0.27
0.64
1.16
224
154
20.9
28.6
3.2
3.5
289
211
-0.40
-0.29
0.75
0.96
433 ± 46
30.5 ± 7.3
3.3 ± 0.2
418 ± 7 4
-0.59 ± 0. 16
0.88 ±0.13
NA
Intron 4 splice site
15
16
Q344ter
18
19
Normals
Mean ± SD11
NA = not available.
* Parameters of the P3 model fit to the rod-isolated a-wave intensity series.
f Parameters of the Naka-Rushton curve fit to rod-isolated b-vvave intensity series.
+ Visit in 1985.
§ Visit in 1992.
" n = 8 for a-wave; n = 57 for b-wave.
Q344ter mutations. Serial measurements in one patient with the Q64ter mutation suggested vulnerability
of the midperiphery with disease progression, and this
is consistent with the findings in more affected
members of this family who had midperipheral scotomas and retained central and peripheral patches of
rod function. A comparison of patients with the mildest phenotypes in the three genotypes indicates that,
on average, patients with the Q64ter mutation had the
greatest degree of rod sensitivity loss and lowest levels
of measurable rhodopsin, whereas those with the
Q344ter mutation were intermediate, and splice site
mutation patients had the least sensitivity loss and
some of the higher levels of rhodopsin measured. The
rod sensitivity losses in all patients were consistent
with decreased probability of light absorption from
the reduced levels of rhodopsin, as has been found in
other rhodopsin mutations.1013 Thus, when dark
adapted, these patients did not appear to have substantial quantities of photolyzed pigment products
acting as a source of equivalent light in the photoreceptors, as has been proposed to occur in some forms
of RP.33
Rod ERG a-wave and b-wave analyses have been
applied to patients with RP of unknown geno21.24.27,29.34-36
but the present study is the first to
type
use this approach to interpret the waveforms from patients with rhodopsin mutations. The a-wave analysis
in our patients showed that the maximum receptor
response (Rmax) was reduced compared to the normal
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mean result, and a-wave sensitivity (a) fell within our
normal limits. The rod b-wave results showed varying
amounts of reduction of Vmax and abnormalities in K.
This ensemble of ERG findings is consistent with certain hypothesized underlying disease mechanisms, but
not with others. For example, a disease process affecting the sensitivity of all rods across the retina in the
same way, such as a decreased number of rhodopsin
molecules in otherwise normal rods,7'8 would not explain the reduced a-wave and b-wave maximum responses and normal a-wave sensitivity.24'29'37 A retina
with well-functioning rods interspersed with nonfunctional receptors could lead to ERG findings such as we
observed in our patients.2429'37 It is also possible that
the disease mechanism is more complex, and partially
functional receptors may be contributing subnormally
to the full-field response.29'37
Rod dark adaptation has been found to be abnormal in many different mutations of the rhodopsin
gene that cause adRP, and kinetic analyses of the results have shown there can be specific abnormalities in
the different genotypes.38 Analyses of rod dark adaptation data in the present study indicate there are very
different mechanisms of dysfunction in the three genotypes. The approximately normal time course of rod
dark adaptation in patients with the Q64ter mutation
suggests that this function is essentially mediated by
wild-type protein. However, the analysis indicates that
at least one of the parameters, k32, is abnormal, and by
similar amounts in both P5 and P7 (Table 2). The in-
2531
Stop Codon Rhodopsin Mutations and RP
400
functional abnormalities (on the basis of this model)
may thus be associated with relatively slow reactions,
such as the binding of arrestin to bleached rhodopsin,
and the reduction and removal of the aU-trans retinal
from the binding site. It has been suggested that the
photoproducts involved in these reactions may play a
role in setting the sensitivity of the rod.34'40"42 The
rates of interconversions of one or more of them may
depend on the integrity of the carboxy terminal region
of rhodopsin.45 In the splice site mutation, this region
of the molecule is likely to be abnormal. Interestingly,
both in patients with this mutation and in those with
the Q344ter mutation, the value of S2s;ll, the half-saturating concentration of S2, is found to be about twice
as large as normal, implying that the factor within the
outer segment that limits the rate of this reaction after
intense bleaching1432 is present in abnormally high
amounts relative to those of the expressed rhodopsin.
Rod dark adaptometry in patients with the
Q344ter mutation also showed a prolonged recovery
of sensitivity, but this differed quantitatively from the
abnormality in the splice site mutation. As expected
from the extended period required for complete sensitivity recovery after a 99% bleach (Fig. 4), the kinetic
Q64ter
300
200
LU 100
Q
Q_
'85i
LU
^
400
'92
o • SPLICE SITE
A • Q344ter
cb
300
200
100
°-2
-1
I I 0T
1
STIMULUS ENERGY [log scot td s]
40 F
FIGURE 7. ERG b-wave amplitude as a function of stimulus
energy for normal subjects and the patients. {Top) Results
from P5 (unfilled squares) and two visits for P7 (filled
squares), both of whom have the Q64ter mutation. (Bottom)
Results from PI 5 (unfilled circles) and PI 6 (filled circles)
with the splice site mutation, and PI 8 (unfilled triangles) and
PI 9 (filled triangles) with the Q344ter mutation. Solid
curves are the fits of the Naka-Rushton function to the patient data; arrows with symbols denote K for the patients.
Dashed curve is a mean normal function; error bar on vertical axis is mean normal V,nax — 2 SD; bar on horizontal axis is
mean normal K ± 2 SD.
crease in k32, which is not seen in the splice site and
Q344ter mutations, suggests the possibility of some
factor that interferes with the control of rod sensitivity, at least during the later stages of recovery.39
In patients with the intron 4 splice site mutation,
unlike the Q64ter mutation, there is prolonged recovery of sensitivity, which is reflected by the low values
found in the kinetic analysis (Table 2) for k34, the parameter that characterizes the last stage in the recovery of sensitivity (and is loosely identifiable with the
regeneration of photo-activatable rhodopsin). Values
were also abnormal for the other parameters associated primarily with the later stages of recovery. The
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100
80
60
40
20
% RHODOPSIN
FIGURE 8. Relationship between rod sensitivity loss and rhodopsin levels in P5 and P7, who carry the Q64ter mutation,
and PI 5 and PI6, who carry the splice site mutation. Data
from P5 and P7 are mean values for the central 10 X 10°
rectangle of the measurement area; data points for PI 5 and
PI6 are individual values from within the measurement
area. The solid line describes the expected relationship if
rod sensitivity loss was caused solely by the decreased probability of light absorption resulting from reduced levels of
rhodopsin. Symbols as in Figure 7.
2532
Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5
analysis yielded values for k23 and k34 that were lower
than those for patients with the splice site mutation
(Table 2). The prolonged desensitization of the rod
after bleaching may relate to defective reactions in the
visual cycle of this mutant opsin because of the altered
carboxy terminus of the molecule. 43 ' 44
The relationship between the results of noninvasive tests of visual function and the underlying photoreceptor pathophysiology is complex, but our findings
in the mildly affected patients permit some speculation about the disease mechanisms in these three genotypes. The Q64ter mutation, in theory, would be a
functional null mutation. The measurable rhodopsin
by fundus reflectometry and the rod-mediated function detected by psychophysics and electroretinography in these patients indicate that some rhodopsin
(presumably only wild-type) has been synthesized,
transported to the outer segment, and inserted into
the disk membrane, and phototransduction does occur. A simple quantal catch model based on 50% of the
rhodopsin molecules in each rod, which was used to
explain the test results in other putative null mutations, 7 does not explain fully the results of the most
mildly affected patients with the Q64ter mutation. Of
course, even these patients may already be at a later
stage of this progressive retinal degeneration, which
could have started with a 50% reduction in the number
of rhodopsin molecules per rod. The unexpected finding of an abnormality in dark adaptation suggests that
some factor, such as partial expression of the mutant
protein or some aspect of the degenerative process of
the disease, may be interfering with recovery of sensitivity of the wild-type protein after light activation.
The intron 4 splice site mutation was also hypothesized to be a null allele based on examination of one
heterozygote with this genotype whose clinical phenotype and rod function abnormalities were similar to
those of heterozygotes with the E249ter mutation. 7
The finding of as much as 75% of normal rhodopsin
levels in some retinal regions by fundus reflectometry
and the abnormality in rod dark adaptation would not
be expected from rods with only half the normal
amount of wild-type rhodopsin. We speculate that
both wild-type and mutant opsins are synthesized,
transported to the outer segment, and inserted into
the disk membrane and that, at very early stages of the
disease, rod outer segment length and rhodopsin concentration may be normal. The abnormal carboxy terminal region of the mutant rhodopsin molecules
would lead to the abnormal kinetics of recovery of
sensitivity after light activation.
The patients with the Q344ter mutation had rod
dark adaptation results that lead to the speculation
that this mutant opsin, like the splice site mutant, is
synthesized and transported to the outer segment,
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where it causes a specific abnormality in the kinetics of
recovery of rod sensitivity after light activation due to
the truncation at the carboxy terminal region. Lending support to our hypothesis that this mutant opsin
may reach the rod outer segment are the results of an
investigation of the biochemical phenotype of the
Q344ter mutant rhodopsin in vitro showing that this
mutant was synthesized, regenerated with 11-cis retinal, and transported to the plasma membrane. 9
The steps leading from the different types of rod
dysfunction in the Q64ter, splice site, and Q344ter
mutations to rod cell death are unknown. Further progress in the elucidation of the exact mechanisms of
dysfunction and cell death of rod photoreceptors resulting from mutations in the rhodopsin gene will require the use of a number of different approaches,
such as studies of the mutant opsins in vitro, 9 ' 4546 of
transgenic animals,47"50 and of donor retinas from patients with known genotypes. 51
An important issue concerning the genetic counseling of patients with rhodopsin gene mutations
arises from the findings in this study. With this report,
there are now five rhodopsin mutations in which some
heterozygotes have been described as having a normal
ophthalmoscopic appearance and relatively mild retinal functional abnormalities: Q344ter, 10 P23H, 13
E249ter, 7 intron 4 splice site (ref. 7 and present
study), and Q64ter (present study). Four of these five
rhodopsin mutations have been associated with adRP;
the exception is the E249ter mutation, in which heterozygotes were considered carriers of arRP. 7 Until we
learn more about the basis for variation in disease expression in RP, caution dictates that all clinically unaffected heterozygotes with rhodopsin gene mutations
should be counseled as if they have adRP. Even if not
destined for severe visual loss themselves, they should
be told of the 50-50 chance of having children who
could express a more severe form of the disease; and,
considering the family with the E249ter mutation, 7
they should be made aware of the chances of producing a homozygote with a consanguineous marriage.
Increasing recognition of the wide spectrum of disease
expression in different genotypes of retinal degeneration makes the determination of the basis of this variation a topic of clinical and scientific importance
warranting further study.52'53
Key Words
null mutation, retinitis pigmentosa, rhodopsin, rod photoreceptor, stop codon
Acknowledgments
The authors thank Mrs. D. Slaughter, Ms. K. Stewart, and
Mrs. B. Koernig for coordinating this study; Dr. X. Sun and
Stop Codon Rhodopsin Mutations and RP
2533
13. Kemp CM, Jacobson SG, Roman AJ, Sung C-H, Nathans J. Abnormal rod dark adaptation in autosomal
dominant retinitis pigmentosa with pro-23-his rhodopsin mutation. Am f Ophthalmol. 1992; 113:165174.
14. Lamb TD. The involvement of rod photoreceptors in
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