Retinal Structure and Function in Achromatopsia Implications for Gene Therapy

ArticleinOphthalmology 121(1) · October 2013with69 Reads
Impact Factor: 6.14 · DOI: 10.1016/j.ophtha.2013.08.017 · Source: PubMed
Abstract

To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy. Cross-sectional study. Forty subjects with ACHM. All subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (1) continuous inner segment ellipsoid (ISe), (2) ISe disruption, (3) ISe absence, (4) presence of a hyporeflective zone (HRZ), and (5) outer retinal atrophy including retinal pigment epithelial loss. Foveal and outer nuclear layer (ONL) thickness was measured and presence of hypoplasia determined. Photoreceptor appearance on SD-OCT imaging, foveal and ONL thickness, presence of foveal hypoplasia, retinal sensitivity and fixation stability, and association of these parameters with age and genotype. Forty subjects with a mean age of 24.9 years (range, 6-52 years) were included. Disease-causing variants were found in CNGA3 (n = 18), CNGB3 (n = 15), GNAT2 (n = 4), and PDE6C (n = 1). No variants were found in 2 individuals. In all, 22.5% of subjects had a continuous ISe layer at the fovea, 27.5% had ISe disruption, 20% had an absent ISe layer, 22.5% had an HRZ, and 7.5% had outer retinal atrophy. No significant differences in age (P = 0.77), mean retinal sensitivity (P = 0.21), or fixation stability (P = 0.34) across the 5 SD-OCT categories were evident. No correlation was found between age and foveal thickness (P = 0.84) or between age and foveal ONL thickness (P = 0.12). The lack of a clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore, the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring the trials' impact. The authors have no proprietary or commercial interest in any of the materials discussed in this article.

Full-text

Available from: Michel Michaelides, Jan 03, 2014
Retinal Structure and Function in
Achromatopsia
Implications for Gene Therapy
Venki Sundaram, FRCOphth,
1,2
Caroline Wilde, MBBS,
1
Jonathan Aboshiha, FRCOphth,
1,2
Jill Cowing, PhD,
1
Colin Han,
3
Christopher S. Langlo,
4
Ravinder Chana, MSc,
1,2
Alice E. Davidson, PhD,
1,2
Panagiotis I. Sergouniotis, MD, PhD,
1
James W. Bainbridge, PhD, FRCOphth,
1,2
Robin R. Ali, PhD,
1
Alfredo Dubra, PhD,
5,6
Gary Rubin, PhD,
1
Andrew R. Webster, MD, FRCOphth,
1,2
Anthony T. Moore, FRCOphth,
1,2
Marko Nardini, PhD,
1
Joseph Carroll, PhD,
4,5,6
Michel Michaelides, MD, FRCOphth
1,2
Purpose: To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical
trials of gene therapy.
Design: Cross-sectional study.
Participants: Forty subjects with ACHM.
Methods: All subjects underwent spectral domain optical coherence tomography (SD-OCT), micr operimetry,
and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (1) continuous
inner segment ellipsoid (ISe), (2) ISe disruption, (3) ISe absence, (4) presen ce of a hyporeective zone (HRZ ), and
(5) outer retinal atrophy including retina l pigment epithelial loss. Foveal and outer nuclear layer (ONL) thickness
was measured and presence of hypoplasia determined.
Main Outcome Measures: Photoreceptor appearance on SD-OCT imaging, foveal and ONL thickness,
presence of foveal hypoplasia, retinal sensitivity and xation stability, and association of these parameters with
age and genotype.
Results: Forty subjects with a mean age of 24.9 years (range, 6e52 years) were included. Disease-causing
variants were found in CNGA3 (n [ 18) , CNGB3 (n ¼ 15), GNAT2 (n ¼ 4), and PDE6C (n ¼ 1). No variants were
found in 2 individuals. In all, 22.5% of subjects had a continuous ISe layer at the fovea, 27.5% had ISe disruption,
20% had an absent ISe layer, 22.5% had an HRZ, and 7.5% had outer retinal atrophy. No signicant differences in
age (P ¼ 0.77), mean retinal sensitivity (P ¼ 0.21), or xation stability (P ¼ 0.34) across the 5 SD- OCT categories were
evident. No correlation was found between age and foveal thickness (P ¼ 0.84) or between age and foveal ONL
thickness (P ¼ 0.12).
Conclusions: The lack of a clear association of disruption of retinal structure or function in ACHM with age
suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than
previously indicated. Therefore, the potential benet for a given subject is likely to be better predicted by speci c
measurement of photo receptor structure rather than simply by age. The ability to directly assess cone photo-
receptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting
subjects for future trials and measuring the trials impac t. Ophthalmology 2014;121:234-245 ª 2014 by the
American Academy of Ophthalmology.
Achromatopsia (ACHM) is a cone dysfunction syndrome
with an incidence of approximately 1 in 30 000, which pres-
ents at birth or early infancy.
1
It is characterized by marked
photophobia and nystagmus, reduced visual acuity (20/120
to 20/200), very poor or absent color vision, and absent
cone electroretinogram responses, with normal rod function.
Fundus examination is usually normal, although retinal
pigment epithelial (RPE) disturbance and atrophy may be
present. Mutations in 5 genes have been identied in
ACHM: CNGA3, CNGB3, GNAT2, PDE6C,andPDE6H,all
of which encode components of the cone phototransduction
cascade.
2e5
CNGA3 and CNGB3 encode the
a
and
b
subunits of the cGMP-gated cation channel, respectively, and
account for approximately 80% of cases of ACHM.
1,2
Sequence variants in GNAT2, PDE6C,andPDE6H are
uncommon causes of ACHM, each accounting for <2% of
patients, and encode the
a
-subunit of transducin and the
a
and
g
subunits of cGMP phosphodiesterase, respectively.
3e5
There have bee n several optical coherence tomography
(OCT)ebased studies that have investigated outer retinal
234 2014 by the American Academy of Ophthalmology ISSN 0161-6420/14/$ - see front matter
Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ophtha.2013.08.017
Page 1
architecture and foveal morphology in ACHM.
6e9
The
macular appearances described include normal lamination,
variable degrees of disruption of the hyperreective photo-
receptor bands (known as either the inner segment/outer
segment [OS] junction or inner segment ellipsoid [ISe]), an
optically empty cavity or hyporeective zone (HRZ), and
complete outer retinal and RPE loss.
6e9
There are signi-
cant limitations to these studies, including the fact that
subjects wer e not genotyped in all cases, and many relied on
qualitative metrics to analyze the OCT images. In addition,
there are conicting data on progression and the presence or
absence of age-dependent outer retinal loss, with Thiadens
et al
6
and Thomas et al
7
suggesting age-associated
progression, whereas Genead et al
8
provided evidence that
cone loss is not age dependent. These inconsistencies, and
the fact that several groups around the world are in
preparation for gene replacement clinical trials, makes it
critical to elucidate the progressive nature (and thus the
therapeutic window) in ACHM in a genotype-dependent
fashion.
Several studies have shown that gene therapy can be
effective in restoring cone function in multiple animal models
of ACHM.
10e14
In a Cngb3
/
mouse model, subretinal gene
delivery resulted in restoration of electrophysiologic function
to near normal levels and signicantly improved visual
behavior,
13
with larger canine models of CNGB3-associated
disease also showing increased electrophysiologic responses
and improvements in navigational ability after gene
replacement therapy.
14
In anticipation of the imminent
human gene therapy trials for ACHM, we sought to
characterize the relationship between retinal structure and
function in a large number of molecularly proven subjects.
This information is important to help identify the most
suitable candidates for therapy, to determine the optimal
timing for intervention, and to measure its impact using
appropriate outcome measures.
Methods
Subjects
Forty subjects with a clinical diagnosis of ACHM were included in
this study. Ten additional subjects with normal vision were
recruited. The protocol of the study adhered to the tenets of the
Declaration of Helsinki, was approved by the local Ethics
Committees of Moorelds Eye Hospital and the Medical College
of Wisconsin, and was performed with the informed consent of all
subjects.
Clinical Assessments
All subjects underwent a clinical history and detailed ocular
examination, including best-corrected visual acuity (BCVA) using
an Early Treatment Diabetic Retinopathy Study chart, reading
acuity using the MNRead chart, contrast sensitivity assessment
using the Pelli-Robson chart at 1 m, color vision testing (Ishihara
and Hardy Rand Rittler pseudoisochromatic plates), color fundus
photography, spectral domain OCT (SD-OCT), and micro-
perimetry (MP).
On the basis of their fundus appearance on color fundus
photography, each subject was assigned to 1 of 3 categories: (1) no
RPE disturbance, (2) RPE disturbance, or (3) atrophy.
Figure 1. Representative images of the 5 optical coherence tomography
phenotypes. Subjects were graded into 1 of 5 categories: (i) continuous
inner segment ellipsoid (ISe) band, (ii) ISe disruption, (iii) ISe absence,
(iv) hyporeective zone present, and (v) outer retinal atrophy. Scale bar,
200
m
m.
Sundaram et al
Retinal Structure and Function in Achromatopsia
235
Page 2
SD-OCT
For all subjects (80 eyes of 40 subjects), after pupillary dilation,
line and volume scans were obtained using a Spectralis SD-OCT
(Heidelberg Engineering, Heidelberg, Germany). The volume
acquisition protocol consisted of 49 B-scans (124
m
m between
scans; 2020
), with Automatic Real Time eye tracking used when
possible. The lateral scale of each image was estimated using the
axial length data obtained from the Zeiss IOL Master (Carl Zeiss
Meditec, Jena Germany).
Qualitative Assessment of Foveal Morphology. Foveal struc-
ture on SD-OCT images was graded into 1 of 5 categories (Fig 1):
(1) continuous ISe, (2) ISe disruption, (3) ISe absence, (4) presence
of an HRZ, or (5) outer retinal atrophy, including RPE loss. The
presence/absence of foveal hypoplasia was also noted, dened as
the persistence of 1 inner retinal layers (outer plexiform layer,
inner nuclear layer, inner plexiform layer, or ganglion cell layer)
through the fovea. Figure 2 shows examples of the varying
degrees of foveal hypoplasia observed in the subjects examined
herein. Consensus grading was established by 3 independent
examiners (V.S., J.C., and M.M.).
Quantitative Analysis of Photoreceptor Structure on SD-
OCT. We used a method that was conceptually similar to that
described by Hood et al
15
to analyze the intensity of the ISe and
external limiting membrane (ELM) bands, although there are
a number of differences. First, our images were transformed into a
linear display using a transform provided by the manufacturer. This
is a critical correction to apply, because the native visualization of
SD-OCT images on a logarithmic scale misrepresents the real
differences in reectivity (Fig 3). Second, we assessed layer
intensity at only 2 specic retinal locations, 1 and 1.5 mm temporal
to the fovea (because of the outer retinal disruption in many
subjects, 1 mm was the closest eccentricity we could measure in all
subjects). Finally, the procedure used to measure layer intensity
was different. We generated longitudinal reectivity proles
16
at
the 1- and 1.5-mm locations, and each longitudinal reectivity
prole was 5 pixels in width (Fig 3). Hood et al
15
dened a local
region surrounding a specic segment of the ISe as extending
275
m
m to either side of the ISe segment and extending axially
between the Bruchs membrane/choroid interface and the posterior
border of the retinal nerve ber layer.
15
Because other posterior
layers may be altered because of the disruption in cone structure,
including the outer nuclear layer (ONL) and Henle ber layer, or
the layers posterior to the ISe that are thought to originate from
interactions between the photoreceptors and RPE,
17
we used
a local region restricted to the retinal ganglion cell layer and
inner plexiform layer. This is indicated by the horizontal arrows in
Figure 3. We then measured the peak image intensity at the ELM
and ISe (labeled in Fig 3), and the relative intensity of the ISe (or
ELM) was taken as the ISe (or ELM) peak intensity divided by the
average intensity in the local region, generating the ISe (or ELM)
intensity ratio used for analysis.
Figure 2. Representative examples of varying degrees of foveal hypoplasia.
Foveal hypoplasia was dened here as the persistence of 1 inner retinal
layers (outer plexiform layer, inner nuclear layer, inner plexiform layer, or
ganglion cell layer) through the foveal center. Normal retinal anatomy (top
panel) shows complete excavation of the inner retinal layers at the fovea,
resulting in the characteristic pit. However, in a number of conditions
(such as retinopathy of prematurity and albinism), this process is impaired
(foveal hypoplasia), resulting in retinas in which the inner retinal layers
persist at the fovea. Interestingly, this can also be seen in achromatopsia.
The 3 lower panels show examples of varying degrees of foveal hypoplasia
in patients with achromatopsia, in whom the fovea contains inner retinal
layers as opposed to complete excavation of these layers. Twenty-one of the
40 subjects (52.5%) had foveal hypoplasia, although it was not possible to
assess hypoplasia in 2 subjects because of severe foveal atrophy. There was
no difference in age, contrast sensitivity, retinal sensitivity, or xation
stability between subjects with or without foveal hypoplasia (see text).
Scale bar, 200
m
m. ND ¼ no data.
<
Ophthalmology Volume 121, Number 1, January 2014
236
Page 3
In addition to examining the ELM and inner segment/OS
intensity, we measured the total retinal thickness (internal limiting
membrane to RPE distance) and ONL thickness (ILM to ELM
distance) at the fovea. In cases of foveal hypoplasia, the distance
between the posterior outer plexiform layer boundary and the ELM
was taken as the ONL thickness. All thickness measurements were
conducted by a single observer (C.H.) using ImageJ software
(National Institutes of Health, Bethesda, MD).
Microperimetry
MP was performed on both eyes of all subjects using the MP-1
microperimeter (Nidek Technologies, Padova, Italy). Specic
details can be found in the online only material (Appendix 1,
available at http://aaojournal.org). Fixation stability was assessed
using the bivariate contour ellipse area (BCEA) which represents
an area in degrees where 68% of xation points are located;
18
this value is reported by the Nidek software.
Molecular Genetic Testing
Conventional direct Sanger sequencing of exons and exon-intron
boundaries of CNGA3, CNGB3, GNAT2 , and PDE6C was under-
taken using previously published methods.
2e4
Subjects 39 and 40
also underwent screening of exons and exon-intron boundaries of
PDE6H.
5
Statistical Analysis
Normality of data was assessed by evaluating the shape of histo-
gram plots, with age, BCVA, contrast sensitivity, and reading
acuity considered to be normally distributed. Intereye correlations
for all parameters were assessed using Pearson or Spearman
correlation analysis where appropriate. The left eye was arbitrarily
selected for further analysis, and differences in parameters between
SD-OCT categories, fundus appearance category, and genotype
were assessed using 1-way analysis of variance or the Kruskal-
Wallis test where appropriate. Differences in parameters between
subjects with or without foveal hypoplasia were assessed using
either an independent samples t-test or ManneWhitney U test
where appropriate.
Results
Twenty male and 20 female subjects with a mean age of 24.9 years
(range, 6e52 years) were included (Tables 1 and 2). Mean BCVA
was 0.92 logarithm of the minimum angle of resolution (range,
0.72e1.32), mean contrast sensitivity was 1.16 logCS (range,
0.50e1.55), and mean reading acuity was 0.76 logarithm
minimum angle of resolution (range, 0.5e1.32; Table 2). There
was no correlation between age and (1) BCVA (r ¼ 0.18; P ¼
0.27; Fig 4), (2) contrast sensitivity (r ¼0.27; P ¼ 0.09), or
(3) reading acuity (r ¼ 0.29; P ¼ 0.07).
All subjects were able to read the Ishihara test plate, but were
unable to read any subsequent plates or to correctly identify any of
the Hardy Rand Rittler test plates.
Fundus examination revealed no evidence of macular RPE
disturbance in 11 subjects (mean age, 19.5 years; range, 6e33 years)
RPE disturbance in 20 subjects (mean age, 27.5 years; range, 12e52
years), and well-circumscribed macular atrophy in 9 subjects (mean
age, 25.9 years; range, 11e43 years; Table 2). There was no
difference in mean age, BCVA, contrast sensitivity, reading
acuity, retinal sensitivity, or bivariate contour ellipse area between
these 3 groups (Table 3, available at http://aaojournal.org).
Figure 3. Longitudinal reectivity prole assessment of photoreceptor integrity on spectral domain optical coherence tomography (SD-OCT). The top
image is the native display of the image from the SD-OCT, whereas the lower image is a linear display of the actual image intensity. Note that in the linear
(raw) SD-OCT image, greater differences in layer intensity get compressed when visualized on a logarithmic display. Moreover, the logarithmic transform
will increase the widths of the hyperreective bands being measured.
17
This highlights the need to use raw SD-OCT data when making quantitative
measures of layer intensity. Vertical lines in each image indicate the location of the longitudinal reectivity prole (LRP) shown to the right of each
image. Short horizontal arrows dene the boundaries of the local region used to normalize the inner segment ellipsoid (ISe) or external limiting
membrane (ELM) intensity. a.u. ¼ arbitrary units.
Sundaram et al
Retinal Structure and Function in Achromatopsia
237
Page 4
Table 1. Summary of Retinal Structure Assessed with Spectral Domain-Optical Coherence Tomography in 40 Patients with Achromatopsia
Pt
No.
Age
(yr) Sex
Axial
Length
(mm) Gene Allele 1/Allele 2
SD-OCT
Category
y
Foveal
Hypoplasia
ELM
Ratio
ISe
Ratio
1
mm
1.5
mm
1
mm
1.5
mm
1 7 M 23.93 CNGA3 c.1641C>A-p.Phe547Leu / c.1641C>A-p.Phe547Leu 3 Y 0.32 0.41 1.2 1.16
2 10 M 21.27 CNGA3 c.1642G>A-p.Gly548Arg / c.67C>T-p.Arg23Ter 1 Y 0.43 0.38 1.2 1.22
3 11 M 23.43 CNGA3 c.485A>T-p.Asp162Val / c.485A>T-p.Asp162Val 1 N 0.75 1.18 1.52 1.71
4 11 F 21.25 CNGA3 c.536T>A-p.Val179Asp / c.536T>A-p.Val179Asp 2 N 0.79 1.12 1.19 1.53
5 17 M 22.74 CNGA3 c.1001C>T-p.Ser334Phe / c.1360A>T-p.Lys454Ter 3 Y 0.66 0.57 1.68 1.09
6 19 F 22.49 CNGA3 c.1694C>T-p.Thr565Met / c.661C>T-p.Arg221Ter 2 N 0.83 0.57 2.25 2.26
7 22 F 23.96 CNGA3 c.847C>T-p.Arg283Trp / c.1279C>T-p.Arg427Cys 2 N 0.62 0.92 1.63 1.04
8 22 F 24.58 CNGA3 c.848G>A-p.Arg283Gln / c.667C>T-p.Arg223Trp 4 Y 0.66 0.64 1.66 1.73
9 24 M 24.57 CNGA3 c.848G>A-p.Arg283Gln / c.667C>T-p.Arg223Trp 2 Y 0.94 0.65 1.88 2
10 25 F 22.77 CNGA3 c.1315C>T-p.Arg439Trp / c.1315C>T-p.Arg439Trp 5 N/A 0.76 1.03 1.32 2.24
11 27 F 29.26 CNGA3 c.661C>T-p.Arg221Ter / c.661C>T-p.Arg221Ter 3 Y 0.76 0.55 2.33 2.41
12 28 F 25.51 CNGA3 c.848G>A-p.Arg283Gln / c.667C>T-p.Arg223Trp 4 Y 0.76 0.79 1.73 1.61
13 29 F 22.40 CNGA3 c.661C
>T-p.Arg221Ter / c.848G>A-p.Arg283Gln 2 N 0.64 0.88 1.11 2.67
14 31 M 23.18 CNGA3 c.848G>A-p.Arg283Gln / c.667C>T-p.Arg223Trp 4 Y 0.54 0.77 1.74 1.59
15 32 F 25.52 CNGA3 c.1641C>A-p.Phe547Leu / c.1641C>A-p.Phe547Leu 2 Y 0.61 0.42 1.75 1.15
16 34 F 24.38 CNGA3 c.1443-1444insC-p.Ile482His fs*6 / c.1706G>A-
p.Arg569His
2 Y 0.55 0.56 1.02 1.21
17 35 M 28.06 CNGA3 c.661C>T-p.Arg221Ter / c.848G>A-p.Arg282Gln 2 N 0.52 0.61 1.99 2.67
18 49 F 24.90 CNGA3 c.67C>T-p.Arg23Ter / c.67C>T-p.Arg23Ter 4 Y 0.69 0.64 1.84 1.27
19 6 M 21.02 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1578þ1G>A - Splice
defect
1 N 0.51 0.73 0.83 1.08
20 11 M 23.63 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
4 Y 0.73 1.05 1.73 2.06
21 11 F 21.21 CNGB3 c.595delG-p.Glu199Ser fs*3 / c.1148delC-p.Thr383Ile
fs*13
2 Y 0.57 0.56 1.28 1.73
22 12 F 23.28 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1006G>T-
p.Glu336Ter
3 Y 0.93 0.83 2.07 1.75
23 12 F 22.24 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
2 Y 0.6 0.49 1.61 1.52
24 13 M 23.42 CNGB3 c.595delG-p.Glu199Ser fs*3 / c.1148delC-p.Thr383Ile
fs*13
1 N 0.44 0.35 1.28 1.19
25 17 F 22.41 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
3 N 0.82 0.76 1.16 1.11
26 18 M 23.51 CNGB3 c.595delG-p.Glu199Ser fs*3 / c.1148delC-p.Thr383Ile
fs*13
4 Y 0.65 0.86 2.04 1.47
27 19 M 22.41 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
4 Y 1.4 0.57 1.96 1.01
28 23 M 23.67 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
5 Y 0.62 0.54 1.55 1.59
29 24 M 22.65 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.607-608insT-
p.Arg203Leu fs*3
1 N 0.54 0.57 0.8 0.67
30 27 M 22.85
CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1853delC-p.Thr618Ile
fs*2
5 N/A 0.48 0.43 2.23 3.91
31 33 F 25.91 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
2 Y 0.53 0.66 1.12 1.26
32 33 F 20.80 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
1 N 0.55 0.89 2.18 1.32
33 47 M 24.13 CNGB3 c.1148delC-p.Thr383Ile fs*13 / c.1148delC-p.Thr383Ile
fs*13
4 N 0.76 0.81 2.34 1.7
34 29 M 22.94 GNAT2 c.843-844insAGTC-p.His282Ser fs*11 / c.843-
844insAGTC-p.His282Ser fs*11
4 N 0.98 0.93 4.65 4.96
35 43 M 23.55 GNAT2 c.843-844insAGTC-p.His282Ser fs*11 / c.843-
844insAGTC-p.His282Ser fs*11
1 N 0.74 0.5 2.68 3.15
36 49 F 23.41 GNAT2 c.843-844insAGTC-p.His282Ser fs*11 / c.843-
844insAGTC-p.His282Ser fs*11
1 N 0.65 0.46 3.47 3.61
37 52 M 24.61 GNAT2 c.843-844insAGTC-p.His282Ser fs*11 / c.843-
844insAGTC-p.His282Ser fs*11
1 N 0.58 0.64 1.99 2.57
38 43 M 27.45 PDE6C c.304C>T-p.Arg102Trp / c.304C>T-p.Arg102Trp 3 N 0.58 0.87 0.93 2.01
39 19 F 22.28 ? ND 3 Y 0.47 0.55 1.29 1.04
40 23 F 23.05 ? ND 3 Y 0.5 0.48 1.01 1.45
Ophthalmology Volume 121, Number 1, January 2014
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Molecular Genetics
Eighteen subjects (45%) had mutations in CNGA3 (mean age, 24.1
years; range, 7e49 years), 15 (37.5%) had mutations in CNGB3
(mean age, 20.4 years; range, 6e47 years), 4 (10%) had mutations
in GNAT2 (mean age, 43.3 years; range, 29e52 years), and 1
subject had a mutation in PDE6C (Tables 1, 2, and 4). Seven novel
mutations were found in our group of 40 subjects (Table 4). No
likely disease-associated variants were identied in 2 individuals,
which included screening for PDE6H mutations, in addition to
these 4 genes. Detailed in silico analysis of both previously
described and novel variants is shown in Table 4.
2,19e22
Foveal Morphology
On the basis of SD-OCT imaging, subjects were placed into 1 of 5
categories (Fig 1): (1) 9 subjects (22.5%) had a continuous ISe
layer at the fovea (mean age, 26.8 years; range, 6e52 years); (2)
11 (27.5%) had ISe disruption at the fovea (mean age, 23.8
years; range, 11e35 years); (3) 8 (20%) had an absent ISe layer
at the fovea (mean age, 20.6 years; range, 7e43 years); (4) 9
(22.5%) had a foveal HRZ (mean age, 28.2; range, 11e 49
years); and (5) 3 subjects (7.5%) had evidence of outer retinal
atrophy at the fovea, including RPE loss (mean age, 25 years;
range, 23e27 years; Table 5, available at http://aaojournal.org).
Of the nine subjects with macular atrophy on fundus
examination, 3 subjects were in SD-OCT category 2, 2 in SD-
OCT category 3, 1 in SD-OCT category 4, and 3 subjects in SD-
OCT category 5. The proportion of subjects with any disruption
in cone structure (SD-OCT categories 2e 5) was consistent with
previous studies.
6e8
There were no differences in the age (P ¼ 0.77), BCVA (P ¼
0.44), contrast sensitivity (P ¼ 0.57), or retinal sensitivity (P ¼
0.21) between subjects in the 5 SD-OCT categories; however,
reading acuity was signicantly worse (P ¼ 0.02) in subjects with
no ISe disruption compared with subjects with an HRZ (Table 5).
Figure 5 shows representative SD-OCT images of subjects of
different ages and genotypes, illustrating the variable appearances
within different genotypes and the lack of age dependence on the
integrity of outer retinal architecture.
Foveal hypoplasia was found in 21 subjects (52.5%) (Table 1;
Table 6, available at http://aaojournal.org), with it not being
possible to assess hypoplasia in 2 subjects because of severe
foveal atrophy. Our rate of hypoplasia was slightly lower than 2
previous reports, which used different denitions of hypoplasia
than that used herein.
6,7
There was no signicant difference in
age, contrast sensitivity, retinal sensitivity, or xation stability
between subjects with or without foveal hypoplasia. Surprisingly,
BCVA (P < 0.01) and reading acuity (P < 0.01) were better in
subjects with evidence of foveal hypoplasia compared with those
without (Table 6).
ELM and ISe Intensity Ratios
The mean intensity ratios of the ISe band, measured at 1 and 1.5
mm from the fovea in the 40 ACHM subjects, were 1.73 (range,
0.80e4.65) and 1.82 (range, 0.67e4.96), respectively (Table 1).
These values were signicantly lower compared with 10 controls
with mean ISe ratios of 4.26 (range, 1.84e7.81; P < 0.001) at 1
mm from the fovea and 3.98 at 1.5 mm (range, 2.01e6.70;
P < 0.001) from the fovea, although there was overlap between
the range of intensity ratios observed in ACHM subjects and
controls.
In contrast, the mean intensity ratios of the ELM band,
measured at 1 and 1.5 mm from the fovea in the 40 ACHM
subjects, were 0.66 (range, 0.32e1.40) and 0.68 (range,
0.35e1.18), respectively (Table 1). These values were similar to
those measured in 10 controls, with mean ELM intensity ratios
of 0.69 (range, 0.39e 1.01; P ¼ 0.47) at 1 mm from the fovea
and 0.60 at 1.5 mm (range, 0.39e 0.84; P ¼ 0.43) from the fovea.
No differences were found in ELM or ISe intensity ratios
between CNGA3 and CNGB3 subjects, with the mean ELM
intensity ratios at 1 and 1.5 mm from the fovea being 0.66 and 0.71
in CNGA3 subjects, and 0.68 and 0.68 in CNGB3 subjects,
respectively (P ¼ 0.51 and P ¼ 0.80). The mean ISe intensity
ratios at 1 and 1.5 mm from the fovea were 1.61 and 1.70 in
CNGA3 subjects, and 1.61 and 1.56 in CNGB3 subjects, respec-
tively (P > 0.99 and P ¼ 0.32).
Foveal ONL and Total Retinal Thickness
Mean foveal thickness and ONL thickness at the fovea in ACHM
subjects was 163.6
m
m (range, 62.0e313.2
m
m) and 67.1
m
m
(range, 26.2e110.5
m
m), respectively, which were signicantly
lower than mean foveal thickness (190.4
m
m; range, 136.2e217.0
m
m; P ¼ 0.02) and mean ONL thickness at the fovea (104.9
m
m;
range, 82.9e119.5
m
m; P 0.001) in controls. Again, it is worth
noting that there was overlap between the ACHM subjects and
controls, consistent with the presence of retained cone nuclei in
some ACHM subjects. No correlation was found between age and
either foveal thickness (
r
¼ 0.03; P ¼ 0.84) or foveal ONL
thickness (
r
¼ 0.26; P ¼ 0.12) in subjects with ACHM.
Microperimetry
All 40 subjects underwent MP testing on 2 occasions. There was
no difference in mean retinal sensitivity and xation stability
between eyes, and further analysis was therefore performed using
the left eye only in each subject. No difference in mean retinal
sensitivity or xation stability was found between subjects rst
and second test; the mean of these 2 tests was used for subsequent
analysis.
The mean retinal sensitivity of the group was 16.6 dB (range,
3.1e19.9 dB), and the mean xation stability of the group was
13.5
(range, 1.7e65
), with signicant negative correlations
found between retinal sensitivity and (1) age (
r
¼0.39; P ¼
0.01; Fig 6), (2) BCVA (
r
¼0.44; P < 0.01; Fig 7), and (3)
reading acuity (
r
¼0.55; P < 0.01). Surprisingly, a signicant
correlation was found between lower contrast sensitivity and
both higher retinal sensitivity (
r
¼ 0.35; P ¼ 0.03) and higher
xation stability (
r
¼0.43; P < 0.01). There was no difference
in mean retinal sensitivity (P ¼ 0.21) or xation stability
(P ¼ 0.34) across the 5 SD-OCT categories (Table 5). There was
a signicant correlation between xation stability and BCVA
(
r
¼ 0.43; P < 0.01).
ELM ¼ external limiting membrane; ISe ¼ inner segment ellipsoid; F ¼ female; M ¼ male; N/A ¼ not possible to assess due to presence of outer retinal
atrophy; No ¼ number; ND ¼ no mutation detected; Pt. ¼ patient; SD-OCT ¼ spectral domain optical coherence tomography.
The cDNA is numbered according to Ensembl transcript ID: CNGA3 ENST00000409937; CNGB3 ENST00000320005; GNAT2 ENST00000351050;
PDE6C ENST00000371447, in which þ1 is the A of the translation start codon.
y
1 ¼ continuous ISe; 2 ¼ ISe disruption; 3 ¼ ISe absence; 4 ¼ hyporeective zone; 5 ¼ outer retinal atrophy.
<
Table 1 (cont.)
Sundaram et al
Retinal Structure and Function in Achromatopsia
239
Page 6
Six subjects had a scotoma (0 dB sensitivity in 1 location),
with a mean sensitivity in this group of 11.0 dB (range, 3.1e14.8
dB), and mean xation stability of 12.8
(range, 1.7e24
). The
mean age of these subjects was 40.2 years (range, 25e52 years)
and mean BCVA was 1.01 logarithm of the minimum angle of
resolution (range, 0.8e1.32). Four of these 6 subjects had RPE
disturbance on fundus examination, with the remaining 2 subjects
having macular atrophy. It is of note that variable macular structure
was seen on SD-OCT, with 3 of the 6 subjects having a normal ISe
layer, and 1 subject each having an absent foveal ISe layer, or
HRZ, or outer retinal atrophy.
GenotypeePhenotype Correlation
The vast majority (82.5%) of subjects in our study had either
CNGA3 or CNGB3 mutations. There were no differences between
the subjects with these 2 genotypes in terms of age, BCVA,
contrast sensitivity, reading acuity, or xation stability (Table 7,
available at http://aaojournal.org). However, retinal sensitivity
was signicantly greater in the CNGB3 group than in the
CNGA3 group (18.1 vs 16.1 dB; P ¼ 0.04).
A comparison of the SD-OCT phenotypes and presence of
foveal hypoplasia failed to identify any consistent differences
between the CNGA3 and CNGB3 subjects. Specically, 26.7% of
subjects (n ¼ 4) with CNGB3 mutations had no ISe disruption,
compared with 11.1% (n ¼ 2) with CNGA3 variants; 20% (n ¼ 3)
harboring CNGB3 variants had ISe disruption compared with
44.4% (n ¼ 8) in the CNGA3 group (Table 7). The presence of an
HRZ was similar in CNGA3 (n ¼ 4; 22.5%) and CNGB3 (n ¼ 4;
26.7%) subjects. Outer retinal atrophy was observed in 13.3% (n ¼
2) of CNGB3 subjects compared with 5.5% (n ¼ 1) of CNGA3
subjects. In the CNGA3 group, 61% of subjects (n ¼ 11) had
foveal hypoplasia, compared with 53% of CNGB3 subjects (n ¼
8). Foveal hypoplasia was not present in the 4 subjects with
GNAT2 mutations or in the single PDE6C subject.
On examination, 44.4% of CNGA3 subjects (n ¼ 7) had
a normal fundus appearance, compared with 20% (n ¼ 3) in the
CNGB3 group; 33.3% of CNGA3 subjects (n ¼ 6) had RPE
disturbance compared with 53% of subjects with CNGB3
variants
(n ¼ 8). The percentage of subjects with macular atrophy was
similar in both groups (CNGA3, 22.3% [n ¼ 4]; CNGB3, 27% [n ¼
4]). Of the 6 subjects with a scotoma on MP, 4 had GNAT2 vari-
ants, 1 had PDE6C variants, and 1 had CNGA3 variants. In addi-
tion, BCVA, contrast sensitivity, reading acuity, and mean
sensitivity were lower in the GNAT2 and PDE6C genotypes,
compared with the CNGA3 or CNGB3 groups; however, the mean
age of subjects with either GNAT2 or PDE6C mutations was
considerably higher than the CNGA3/CNGB3 group (Table 7).
Interestingly, 3 out of the 4 subjects with GNAT2 mutations had
an intact ISe layer, despite a relatively low mean retinal
sensitivity of 13.6 dB, with all of these subjects having a central
scotoma on MP.
Discussion
Lack of Age Dependence of Cone Loss
Our cross-sectional study (n ¼ 40) identied no age-
dependent loss of cone structure in subjects with ACHM.
For e xample, we found that cone loss (SD-OCT categories
Table 2. Summary of Clinical Characteristics
Variable Mean (SD) Range Median No. (%)
Age (yr) 24.9 (12.3) 6e52 23.5 d
Visual acuity (logMAR) 0.92 (0.13) 0.72e1.32 0.9 d
Contrast sensitivity (logCS) 1.16 (0.23) 0.50e1.55 1.2 d
Reading acuity (logMAR) 0.76 (0.19) 0.50e1.32 0.73 d
Retinal sensitivity (dB) 16.6 (3.4) 3.1e19.9 17.6 d
BCEA (degrees) 13.5 (13.5) 1.7e65 7.7 d
Genotype
CNGA3 ddd18 (45)
CNGB3 ddd15 (37.5)
GNAT2 ddd4 (10)
PDE6C ddd1 (2.5)
Unknown ddd2 (5)
SD-OCT category*
1 ddd9 (22.5)
2 ddd11 (27.5)
3 ddd8 (20)
4 ddd9 (22.5)
5 ddd3 (7.5)
Foveal hypoplasia
No ddd17 (42.5)
Yes ddd21 (52.5)
Unrecordable ddd2 (5)
Fundus appearance category
y
1 ddd11 (28)
2 ddd20 (50)
3 ddd9 (22)
logMAR ¼ logarithm of the minimum angle of resolution; logCS ¼
logarithm of contrast sensitivity; SD ¼ standard deviation; dB ¼ decibels;
BCEA ¼ bivariate contour ellipse area; SD-OCT ¼ spectral domain
optical coherence tomography; ISe ¼ inner segment ellipsoid; RPE ¼
retinal pigment epithelium.
*SD-OCT category: 1 ¼ continuous ISe; 2 ¼ ISe disruption; 3 ¼ ISe
absence; 4 ¼ hyporeective zone present; 5 ¼ outer retinal atrophy.
y
Fundus appearance category: 1 ¼ no RPE disturbance; 2 ¼ RPE distur-
bance; 3 ¼ Atrophy present.
Figure 4. No signicant decline in visual acuity as a function of age. Forty
subjects with a mean age of 24.9 years (range, 6e52) were included in this
study, with a mean visual acuity of 0.92 (range, 0.72e1.32). There was no
correlation between age and visual acuity (r ¼ 0.18; P ¼ 0.27). Acuity
reported as logarithm minimum angle of resolution (logMAR).
Ophthalmology Volume 121, Number 1, January 2014
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3e5) was evident in approximately 57% of subjects (16/28)
<30 years of age, but in only 33% (4/12) aged >30 years.
Moreover, subjects without ISe disruption (most preserved
cone structure) had the second greatest mean age of t he 5
SD-OCT categories; no reduction in ISe intensity was found
with advancing age. In addition, foveal ONL and total retinal
thickness was signicantly reduced, although we did not nd
an association between retinal thinning and advancing age. In
contrast with our ndings, Thiadens et al
6
reported that cone
loss occurred in 42% of affected individuals (8/19) who were
<30 years of age, with 95% (20/21) >30 years old showing
cone loss on SD-OCT.
6
Thomas et al
7
also reported
age-dependent ONL thinning.
7
One possible explanation
for this discrepancy is the lack of standardization of how
cone loss is measured. Moving forward, it will be
impo rtant to cond uct larger studies with molecularly
proven subjects in which the same anatomic measures are
undertaken.
Table 4. Summary of Potentially Disease-Associated Nonsynonymous Sequence Variants Identied in CNGA3, CNGB3 ,
GNAT2, and PDE6C
Gene
Nucleotide
Alteration
Protein
Alteration Alleles
EVS
Observed
Allele Count
SIFT Tolerance
Index (0e1)
PoyPhen2
HumVar
Score
(0e1)
Blosum 62
Score
(L4 to 11)
Disease
Causing
Previously
Reported
CNGA3 c.848G>A p.Arg283Gln 6 A¼2/
G¼10756
DAMAGING 0.02 PRD 0.996 1 Yes Kohl et al 1998
19
CNGA3 c.661C>T p.Arg221Ter 5 ND NA NA NA Yes Johnson et al 2004
2
CNGA3 c.667C>T p.Arg223Trp 4 ND DAMAGING 0.00 PRD 1.000 3 Yes Wissinger et al
2001
20
CNGA3 c.1641C>A p.Phe547Leu 4 ND DAMAGING 0.05 PRD 0.999 0 Yes Kohl et al 1998
19
CNGA3 c.67C>T p.Arg23Ter 3 ND NA NA NA Yes Johnson et al 2004
2
CNGA3 c.485A>T p.Asp162Val 2 ND TOLERATED 0.6 POS 0.908 3 Possibly Wissinger et al
2001
20
CNGA3 c.536T>A p.Val179Asp 2 ND DAMAGING 0.00 PRD 0.941 3 Yes This study
CNGA3 c.1315C>T p.Arg439Trp 2 ND DAMAGING 0.01 POS 0.901 3 Yes This study
CNGA3 c.1642G>A p.Gly548Arg 1 ND DAMAGNG 0.00 PRD 1.000 2 Yes Johnson et al 2004
2
CNGA3 c.847C>T p.Arg283Trp 1 T¼1/
C¼10757
DAMAGING 0.00 PRD 1.000 3 Yes Kohl et al 1998
19
CNGA3 c.1001C>T p.Ser334Phe 1 ND TOLERATED 1.00 POS 0.744 2 Possibly This study
CNGA3 c.1694C>T p.Thr565Met 1 T¼2/
C¼13004
DAMAGING 0.03 PRD 0.999 1 Yes Wissinger et al
2001
20
CNGA3 c.1360A>T p.Lys454Ter 1 ND NA NA NA Yes This study
CNGA3 c.1279C>T p.Arg427Cys 1 T¼10/
C¼10748
DAMAGING 0.00 PRD 1.000 3 Yes Wissinger et al
2001
20
CNGA3 c.1706G>A p.Arg569His 1 A¼1/
G¼13005
DAMAGING 0.00 PRD 0.991 0 Yes Wissinger et al
2001
20
CNGA3 c.1443-1444insC p.Ile482His fs*6 1 ND NA NA NA Yes Johnson et al 2004
2
CNGB3 c.1148delC p.Thr383Ile
fs*13
23 ND NA NA NA Yes Kohl et al 2000
21
CNGB3 c.595delG p.Glu199Ser
fs*3
3 ND NA NA NA Yes Johnson et al 2004
2
CNGB3 c.1006G>T p.Glu336Ter 1 ND NA NA NA Yes Kohl et al 2000
21
CNGB3 c.607-608insT p.Arg203Leu
fs*3
1 ND NA NA NA Yes This study
CNGB3 c.1853delC p.Thr618Ile
fs*2
1 ND NA NA NA Yes This study
CNGB3 c.1578þ1G>A Splice defect 1 ND NA NA NA Yes Kohl et al 2000
21
GNAT2 c.843-
844insAGTC
p.His282Ser
fs*11
8 ND NA NA NA Yes Aligianis et al
2002
22
PDE6C c.304C>T p.Arg102Trp 2 ND DAMAGING 0.00 PRD 0.999 3 Yes This study
NA ¼ not applicable; ND ¼ not detected; POS ¼possibly damaging; PRD ¼ probably damaging.
The predicted biological effect of nonsynonymous variants identied in CNGA3, CNGB3, GNAT2 and PDE6C were scored for likely pathogenicity using
EVS, SIFT, PolyPhen2, and Blosum62. EVS denotes variants in the Exome Variant Server, NHLBI Exome Sequencing Project, Seattle, WA (available
from: http://snp.gs.washington.edu/EVS/, accessed April, 2013). SIFT (version 4.0.4; http://sift.jcvi.org/, accessed April, 2013) results are reported to be
tolerated if tolerance index 0.05 or damaging if tolerance index <0.05. Polyphen-2 (version 2.1 http://genetics.bwh.harvard.edu/pph2/, accessed April,
2013) appraises mutations qualitatively as benign, POS or PRD based on the models false positive rate. In the Blosum62 (http://www.ncbi.nlm.nih.gov/
Class/FieldGuide/BLOSUM62.txt, accessed April, 2013) substitution matrix score, positive numbers indicate a substitution more likely to be tolerated
evolutionarily and negative numbers suggest the opposite.
The cDNA is numbered according to Ensembl transcript ID: CNGA3 ENST00000409937; CNGB3 ENST00000320005; GNAT2 ENST00000351050;
PDE6C ENST00000371447, in which þ1 is the A of the translation start codon. Mutations in the coding region of each gene and at intron-exon
boundaries are identied.
Sundaram et al
Retinal Structure and Function in Achromatopsia
241
Page 8
Although our study demonstrates that outer retinal
changes do not necessarily occur in a predictable, age-
dependent manner, a recent small (n ¼ 8), longitudinal
study observed progressive changes in retinal morphology
in younger but not older patients.
23
However, bearing in
mind the characteristic phenotypic variability of
inherited retinal disease, longitudinal studies are needed
to examine the progressive nature of ACHM. It is
Figure 5. Variable spectral domain optical coherence tomography (SD-OCT) appearance in subjects of various ages and genotypes. Representative SD-
OCT images of subjects of different ages and genotypes, illustrating the variable appearances within different genotypes and the lack of age dependence
on the integrity of outer retinal architecture.
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important to note that progression does not imply that age
alone should be a principal eligibility criterion for
emerging trials, as different patients likely progress at
differen t rates.
Retinal Function
We identied no correlation of deterioration in BCVA,
contrast sensitivity, or reading acuity with advancing age,
and to the best our knowledge, this is the largest study to
date reporting any potential change in these parameters with
age. We did, however, nd a decline in MP-based retinal
sensitivity with age. A signicant reduction in retinal
sensitivity, determined by mesopic MP, has been reported to
occur in normal subjects with increasing age, with a 1-dB
lower retinal sensitivity found in subjects aged 70 to 75
years compared with those aged 20 to 29 years.
24
In our
study, the decline in sensitivity of 3.1 dB observed
between subje cts <25 years of age (n ¼ 22) and those
>25 years old (n ¼ 18) is greater than that potentially
attributable to age-related decline.
In our cohort of subjects with absent cone function (on
the basis of electrophysiology and psychophysics), it is
presumed that retinal sensitivity detected by mesopic MP
testing is a consequence of retained rod function. This raises
the question as to whether rod function declines in subjects
with ACHM with age and if so, why? If there is no change
in rod function, it remains a possibility that there is residual
central cone function in some subjects, which may deteri-
orate over time. Further investigation is required to deter-
mine which class of photoreceptor(s) is responsible for the
retinal sensitivity detected by mesopic MP in ACHM. For
example, measuring the rate of recovery of retinal sensitivity
using the microperimeter after a full bleach might help to
shed light on this intrigui ng issue.
StructureeFunction Relationships
We identied no clear association between retinal structure
and function, with no differences in BCVA, contrast
sensitivity, retinal sensitivity, or xation stability between
subjects with the various SD-OCT ndings or fundus
changes. This is in keeping with the lack of an association
reported between the presence of an HRZ and visual acuity
in previous studies.
6,7
Surprisingly, subjects without ISe
disruption had a signicantly lower (P ¼ 0.02) reading
acuity compared with subjects with HRZ presence;
however, a statistical difference was not found in any other
functional parameter, suggesting this is not a clinically
signicant observation. No correla tion was found between
ISe intensity and retinal sensitivity, although this is perhaps
to be expected; in the absence of cone function, retinal
sensitivity is likely to be primarily derived from rod function
in ACHM subjects.
We found no differences in contrast sensitivity, retinal
sensitivi ty, or xat ion stability in subjects with or without
foveal hypoplasia; however, surprisingly, signicantly
better BCVA and reading acuity were found in subjects
Figure 6. Negative correlation between age and retinal sensitivity. All 40
subjects underwent microperimetry testing on 2 occasions. There was no
difference in mean retinal sensitivity between both eyes, and further
analysis was therefore performed using the left eye only. No difference in
mean retinal sensitivity was found between subjects rst and second tests,
and the mean of these 2 tests was used for subsequent analysis. The mean
retinal sensitivity of the group was 16.6 decibels (range, 3.1e19.9), with
signicant negative correlation found between retinal sensitivity and age
(
r
¼0.39; P ¼ 0.01). Acuity is reported as logarithm minimum angle of
resolution (logMAR). dB ¼ decibels.
Figure 7. Negative correlation between visual acuity and retinal sensi-
tivity. The mean retinal sensitivity of the cohort was 16.6 decibels (dB)
(range, 3.1e19.9 dB) with signicant negative correlation found between
retinal sensitivity and visual acuity (
r
¼0.44; P < 0.01). logMAR ¼
logarithm minimum angle of resolution.
Sundaram et al
Retinal Structure and Function in Achromatopsia
243
Page 10
with foveal hypoplasia. This is reminiscent of ndings in
albinism, where the absence of a fovea does not necessarily
impair acuity.
25,26
However, a structural grading system for
foveal hypoplasia reported by Thomas et al
27
has suggested
a r elationship between foveal development and acuity. It is
also likely that varying degrees of nystagmus amplitude or
frequency between subjects contribute to determining
BCVA and retinal sensitivity in ACHM and other
disorders.
Implications for Gene Therapy
Gene replacement trials for both CNGA3 and CNGB3 are
planned in the near future. Our ndings of no age depen-
dence of cone loss demonstrate that the potential window of
opportunity for therapeutic intervention in ACHM is wider
than has previously been suggested; subjects with no
evidence of ISe disruption were aged between 6 and 52
years and we found no correlation of cone photoreceptor
disruption or loss with increasing age.
Because this was a cross-sectional study, it has not
assessed whether subjects who have any form of outer
retinal change develop progressive degeneration, and, if so,
how variable the rate of change may be. With respect to
cone photoreceptor structure, we therefore suggest that
candidates should be considered for potential gene therapy
intervention on an individual basis, irrespective of their age.
In addition, we did not observe decreas ed visual function in
subjects with foveal hypoplasia; in fact, signicantly better
BCVA and reading acuity were found in subjects with
foveal hypoplasia, suggesting that foveal hypoplasia per se
should not be an exclusion criterion for potential therapy
trials. In the 9 subjects with no ISe disruption evident on
SD-OCT, their mean ISe intensity ratio was considerably
lower than in healthy controls, illustrating that assessment of
the degree of residual cone structure using this metric may
be useful in determining the suitability of potential trial
participants. Direct visualization of the cone mosaic is
afforded through the use of adaptive optics imaging.
28e30
There is a need to elucidate the relationship between these
various measures of cone structure in ACHM to establish
the most appropriate means to identify suitable patients and
track therapeutic efcacy.
In addition to cone photoreceptor integrity, anothe r factor
likely to inuence the response to gene therapy is the ability
of the visual system to respond to newly acquired input.
Functional magnetic resonance imaging has shown evidence
of visual cortex reorganization in ACHM subjects, with the
area of visual cortex normally active after cone-derived
foveal stimulation being active instead after rod stimuli.
31
Conversely, recovery of cone-driven cortical activity has
been observed in a canine model of ACHM (Gingras G.
Cortical recovery following gene therapy in a canine model
of achromatopsia. Paper presented at: The Vision Sciences
Society Meeting, May 8, 2009; Florida). The extent to
which the visual cortex is able to adapt to and process new
input from cone photoreceptors is an additional consider-
ation likely to inuence the efcacy of gene replacement
therapy.
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Footnotes and Financial Disclosures
Originally received: April 13, 2013.
Final revision: August 9, 2013.
Accepted: August 13, 2013.
Available online: October 20, 2013. Manuscript no. 2013-608.
1
UCL Institute of Ophthalmology, University College London, London,
UK.
2
Moorelds Eye Hospital, London, UK.
3
Summer Program for Undergraduate Research, Medical College of Wis-
consin, Milwaukee, Wisconsin.
4
Department of Cell Biology, Neurobiology & Anatomy, Medical College
of Wisconsin, Milwaukee, Wisconsin.
5
Department of Ophthalmology, Medical College of Wisconsin, Milwau-
kee, Wisconsin.
6
Department of Biophysics, Medical College of Wisconsin, Milwaukee,
Wisconsin.
J.C. and M.M. are considered joint senior authors.
Financial Disclosures:
The authors have no proprietary or commercial interest in any of the
materials discussed in this article.
Supported by grants from the National Institute for Health Research
Biomedical Research Centre at Moorelds Eye Hospital NHS Foundation
Trust and UCL Institute of Ophthalmology, Fight for Sight, Moorelds Eye
Hospital Special Trustees, The Wellcome Trust, Retinitis Pigmentosa
Fighting Blindness, and the Foundation Fighting Blindness (USA). M.M. is
supported by a Foundation Fighting Blindness Career Development Award.
J.W.B. is an NIHR Research Professor. MCW Funding: NIH grants
R01EY017607, P30EY001931, C06RR016511, Foundation Fighting
Blindness, and an unrestricted departmental grant from Research to Prevent
Blindness (RPB). A.D. is the recipient of a Burroughs Wellcome Fund
Career Award at the Scientic interface and a Career Development Award
from RPB.
Correspondence:
Michel Michaelides, MD, FRCOphth, UCL Institute of Ophthalmology,
11-43 Bath Street, London, EC1V 9EL, UK. E-mail: michel.michaelides@
ucl.ac.uk.
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