CNGB3 Achromatopsia with Progressive Loss of Residual Cone Function and Impaired Rod-Mediated Function

Abstract

purpose. CNGB3 encodes the β-subunits of cyclic nucleotide-gated channels in the photoreceptor plasma membrane. CNGB3 mutations cause a channelopathy that results in impaired cone function manifesting achromatopsia. The clinical physiology and phenotype of three affected sisters and three carriers were evaluated in a family with a homozygous CNGB3 mutation and an unrelated male harboring both CNGB3 and CNGA3 mutations.

methods. Index patients were screened for mutations in CNGA3 and CNGB3 by DNA sequencing. Visual examination included acuity, color vision, Goldmann visual fields (GVF), dark-adapted absolute thresholds (DAT), electroretinography, and fundus photography.

results. The three affected sisters were homozygous for a 1-bp deletion (c.1148delC) in CNGB3 that induces a frame shift after Thr383, whereas the carriers were heterozygous for this mutation. The unrelated male carried a heterozygous 8-bp deletion (c.819_826del8bp) in exon 6, as well as a heterozygous base substitution (c.1208G→A) in exon 11 that causes an Arg403Gln exchange. All affected subjects had acuity ranging between 20/200 and 20/400, moderately constricted GVFs, normal DATs, reduced rod b-wave amplitudes, and extinguished photopic b-wave and flicker responses. Rod photoreceptor sensitivity and amplitude, calculated by fitting the rod a-waves by a model of activation of phototransduction were below normal mean. Carriers had mildly decreased acuity (20/25–20/40), normal rod and cone ERGs, and normal color vision. The fundi of the affected subjects showed macular atrophy by middle age, while the carriers showed peripheral RPE granularity in childhood and macular atrophy in late middle age.

conclusions. Foveomacular atrophy can occur in CNGB3-affected subjects, and even heterozygous carriers can exhibit maculopathy. Cone ERG responses in affected subjects are nearly extinguished, but some retain residual function into middle age and then progressively lose even this remnant. Rod responses are impaired in some CNGB3-affected subjects.

Achromatopsia is an autosomal recessive congenital trait characterized by the absence of color discrimination, with reduced visual acuity, nystagmus, and photophobia. ^{1 2} It results from dysfunction or nonfunction of retinal cone photoreceptors ³ and thus far has been associated with mutations in the CNGA3, CNGB3, and GNAT2 genes. ^{4 5 6 7} GNAT2 encodes the α-subunit of the G-protein transducin that couples to the excited visual pigments, whereas CNGA3 and CNGB3 encode the two subunits of the cyclic nucleotide-gated (CNG) channel that represents the light-activated conductance channels located in cone photoreceptors. This puts achromatopsia in the category of a “channelopathy” condition. This particular subtype of CNG channel is present in the plasma membrane of retinal cone outer segments. ^{8 9} RT-PCR experiments showed that at least CNGA3 is also expressed in olfactory sensory neurons, the cortex, and pineal gland ¹⁰ and in some non-neural tissues of the kidney, lung, colon, testis, and heart. ¹¹ CNG channels play an essential role in sensory transduction in photoreceptors. They are directly activated by 3′,5′-cyclic monophosphate (cGMP) at the end of the visual transduction cascade and control the flow of ions across the surface membrane of photoreceptor outer segments in the vertebrate retina. 

The native CNG channels of rod (CNG1) and cone (CNG3) outer segments ^{12 13} consist of α- and β-subunits. α-Subunits by themselves can reconstitute functional channels in heterologous expression systems, whereas β-subunits by themselves cannot. The β-subunits modulate channel properties when coexpressed with their corresponding α-subunits and thereby produce the specific characteristic of native CNG channels. ^{14 15} Both subunits are essential to constitute normal CNG cation channels in the cone outer segment plasma membrane and are required for generation of light-evoked electrical photoresponses in the red-, green-, and blue-sensitive cones. ⁶ Mutations in either the α-subunit (CNGA3 gene) or the β-subunit (CNGB3 gene) can cause achromatopsia. ^{4 5 6}  

We report the physiologic dysfunction in this type of channelopathy from changes in the cone photoreceptor cyclic nucleotide-gated channel. We characterized the clinical physiology of photoreceptor function in two families with two CNGB3 genotypes and report several novel features for this form of achromatopsia. First, several affected CNGB3 individuals showed outright macular atrophy beyond anything previously reported in this condition. Second, several affected individuals retained some cone function into middle age on ERG testing and showed progressive loss over intervals of 6 to 12 years. Third, although the CNGB3 protein subunit is known to be expressed only in cone photoreceptors, some affected individuals demonstrated rod functional abnormalities to various degrees. 

Methods

Subjects

Family A has three affected sisters—A1 (49 years of age), A2 (45 years of age), A3 (40 years of age)—and three obligate carriers—C1 (70 years of age), C2 (9 years of age), and C3 (5 years of age). Family B has one affected male subject B1 (57 years of age). The study complied with the tenets of the Declaration of Helsinki, and we obtained informed consent from all participating subjects, in accordance with the University of Michigan Medical School Institutional Review Board. 

Mutation Screening

DNA was isolated from peripheral blood lymphocytes by standard procedures. Index patients were screened for mutation in CNGA3 and CNGB3 by direct sequencing of PCR products obtained from amplifying the coding exons with primers located in flanking introns or untranslated region sequences, as described previously. ^{4 5}  

Visual Function

Visual examination included visual acuity, color vision, Goldmann kinetic visual fields (GVFs), dark-adapted absolute thresholds, electroretinography, and fundus photography. The GVFs were obtained by using targets V4e, II4e, and I4e on a standard background (10 cd/m²) and subsequently on a dim 1-cd/m² background. Color vision was evaluated with the Farnsworth D-15, the Sloan achromatopsia test, the Ishihara plate test (24-plate edition), and box 8 of the Lanthony New Color Test (NCT; Richmond Products, Albuquerque, NM). Color tests were performed under 200-lux CIE Standard Illuminant C from a MacBeth easel lamp (Corning, Corning, NY). Pupils were fully dilated with topical phenylephrine HCl (10%) and tropicamide (1%). Absolute thresholds after 45 minutes of dark adaptation were measured at fixation and at several locations along the horizontal meridian on a Goldmann-Weekers Dark-Adaptometer (Haag Streit, Bern, Switzerland). 

Clinical ERG

Ganzfeld (full-field) ERGs were recorded with bipolar gold lens electrodes (Diagnosys, Lowell, MA) and proparacaine HCl (0.5%) topical corneal anesthesia, with the common electrode placed on the forehead. ERGs were recorded as described previously. ¹⁶ Briefly, scotopic responses were recorded using 0.5-Hz dim blue (Wratten 47; Eastman Kodak, Rochester, NY) xenon flashes with maximum flash energy of 1.85 log scotopic (sc) td-s. Flashes were attenuated with neutral-density filters over a 3-log unit range. Population normal values were obtained from 50 control subjects. 

Photopic flicker ERGs were elicited with xenon flashes of 2.16 log td-s (PS-22 Photostimulator; Grass Technologies, West Warwick, RI). Harmonic analysis was performed to generate a Fourier series as described previously. ¹⁷ The population normal values were obtained from 40 eyes of 20 control normal subjects. 

Phototransduction a-Wave Modeling

The rod b-wave abnormalities that have been reported could arise downstream of rod photoreceptor function. Consequently, we studied rod photoresponse characteristics of the rod ERG by analysis of scotopic a-wave responses to 1-ms bright photostrobe flashes (model 283; Vivitar, Santa Monica, CA) presented in a Ganzfeld bowl, as previously described. ¹⁶ The maximum flash energy of 2.7 log cd-s/m², or 4.7 log sc td-s, was attenuated over a 3-log-unit range by neutral-density filters. The leading edge of the ERG a-wave response was fitted with the Hood and Birch version ¹⁸ of the Lamb and Pugh ¹⁹ rod model (equation 1)of the biochemical processes involved in the activation of phototransduction.  

\[\mathrm{P}_{3}\ (\mathit{I,\ t})\ {=}\ R_{\mathrm{max}}{\{}1\ {-}\ \mathrm{exp}{[}{-}I\ {\cdot}\ S\ {\cdot}\ (t{-}t_{\mathrm{d}})^{2}{]}{\}}\ \mathrm{for}\ t\ {>}\ t_{\mathrm{d}}\]

where P ₃ is the underlying photoreceptor component, R _max is maximum amplitude, S is sensitivity, and t _d is a brief time delay. ²⁰ The response to the brightest flash was fitted first, allowing R _max and S to be free parameters, and the remaining individual curves for dimmer intensities were fitted, allowing S to vary, but R _max and t _d to be held constant. ^{16 21}  

In the control subjects, cone responses were recorded every 15 seconds across the same intensity range on a rod-saturating background of 3.3 log td, and these responses were computer subtracted from the mixed rod–cone response to obtain the rod response. ²⁰ In the case of CNGB3 achromats, there was no measurable cone a-wave even at the brightest flash (4.4 log photopic td-s). The bright flash a-wave responses were therefore rod-only responses. 

In one carrier, cone a-waves were recorded using the bright flash (maximum flash 4.4 log ph td-s) over a 3-log-unit range after 10 minutes of light adaptation to a 3.3-log-td rod-saturating background, as described previously. ¹⁶ For model fitting to the cone a-wave data, an additional stage of low-pass filtering with time constant τ (equation 2) , was included in equation 1to account for cone outer segment membrane capacitance. ^{22 23}  

\[\mathrm{P}_{3}\ (I,\ t){=}R_{\mathrm{max}}\ {\{}1{-}\ \mathrm{exp}{[}{-}I\ {\cdot}\ S\ {\cdot}\ (t{-}t_{\mathrm{d}})^{2}{]}{\}}{\ast}e^{{-}(t/{\tau})}\]

In equation 2the asterisk (*) denotes convolution, and only the first 10 ms after flash onset are fitted by the model. ^{20 23}  

Results

Mutation Analysis

Family A.

The three affected sisters carried a homozygous 1-bp deletion (c.1148delC) in exon 10 that results in a frameshift downstream of amino acid Thr383 and introduces a novel 12-amino-acid sequence that terminates with a premature stop codon. If expressed, this would yield a truncated mutant protein of only 394 amino acid residues versus the normal 809 amino acid residue protein of the wild-type (WT) channel subunit. 

Family B.

Affected male B1 was a compound heterozygote with an 8 bp-deletion (c.819_826del8) in exon 6 on one allele and an amino acid substitution Arg403Gln in exon 11 on the second CNGB3 allele. The c.819_826del8 mutation induces a frameshift downstream of Pro273 including a tail of 12 novel amino acid residues up to a premature stop codon. Both parents carried one of these two mutations (Table 1) . In addition, subject B1’s father had an amino acid substitution Arg427Cys in exon 7 of CNGA3 that was transmitted to his son. 

Ophthalmoscopy

Both the affected (Fig. 1)and carrier subjects (Fig. 2)had overt retinal structural changes observable by ophthalmoscopy, ranging from minimal to extensive bilateral macular atrophy as shown in Figure 1 . Heavy granularity and pigmentary disruption of the retinal pigment epithelium (RPE) across the entire fundus including the periphery was also observed in the affected individuals. All three carriers, including C3 (5 years of age), showed clinically obvious RPE granularity that was far beyond the spectrum of minimal RPE granularity that some subjects otherwise deemed clinically normal may exhibit. Subject C1 (70-year-old carrier mother in family A) had macular RPE mottling that mirrored her two affected daughters, as shown in Figure 2 . Because of their ages, the carrier parents in family B with the CNGB3 missense mutation were not available for examination. Acuity ranged from 20/200 to 20/400 in the affected subjects, and from 20/25 to 20/40 in the carriers. Refractive error varied from +3.25 to −6.00 D spherical equivalent. 

Goldmann Visual Fields

Visual fields for all CNGB3-affected subjects showed a mild constriction to all three targets under standard background conditions. No central scotomas were detected for any subject with the target sizes used. When the background lighting wasdimmed, the fields expanded by 10° to 20° for all four affected subjects, as is seen with achromatic subjects who have only rod function. 

Color Discrimination Testing

Ishihara Plate Test.

The affected subjects could correctly discriminate and identify numbers on plates beyond the test plate. All four affected subjects (in families A and B) correctly identified two to four plates in addition to the test plate (Table 2 . Subject A1 correctly identified the most digits among the affected individuals. As each plate is otherwise intensity neutral to achromatic rod vision, this result in the four affected subjects indicates residual color hue discrimination by the cone system. Residual color discrimination suggests the presence of a functional receptor in addition to rods in some CNGB3 patients. Blue cone visual signals are known to interact with rod vision and thereby mediate color discrimination beyond achromatic vision. ²⁴ It is unknown whether this residual color vision in patients with mutations in CNGB3 is expressed by blue cones that are phylogenetically separate from red and green cones. The homozygous CNGA3 ^−/− mouse, however, shows no evidence of S- or M-wavelength sensitive cone function. All carriers correctly identified all Ishihara plates and exhibited no evidence that haploinsufficiency had impaired their full color discrimination. 

Farnsworth Dichotomous D-15.

All four affected subjects arranged the colors in the order from brightest to darkest hue and on average followed a scotopic axis, ²⁵ although some variation was found among subjects and on repeat testing of each subject. The affected subjects indicated that the blues were the “brighter colors,” and the reds and purples were “dark” colors; however, none of the affected subjects could actually name any color correctly. None of the carriers showed any errors in the arrangement of the D-15. 

Sloan Achromatopsia Test.

All four CNGB3 affected individuals reported this test to be the easiest, whereas it was difficult for the carriers. The mean match (Table 3)of the affected subjects was comparable to the mean reported for achromats. ²⁶  

Lanthony New Color Test.

This test (Richmond Products) was performed as described in the manual, with the subject viewing box 8 with both eyes. The task is to separate the chips with color from those without color. None of the four CNGB3 achromats could discriminate between the colored and noncolored chips; instead, they all ordered the caps from dark to light while intermingling the colored with the noncolored chips. 

Dark-Adapted Absolute Threshold

Dark-adapted absolute threshold sensitivity of all four affected subjects was within the normal range for macular fixation (CNGB3 subjects: 1.26 log asb ± 0.37 SD; control subjects: mean 1.66 log asb ± 0.66 SD, n = 38) and at 60° in the temporal retina. (CNGB3 subjects: 1.36 ± 0.36 SD; control subjects: mean 1.56 log asb ± 0.16 SD, n = 38). 

ERG Recordings

ERG responses of a representative normal subject, affected subject A2 and carrier C2 are shown in Figure 3A . Affected subjects had highly reduced or negligible light-adapted responses (i.e., amplitudes <5 μV without computer averaging). The 32-Hz flicker responses were diminished in all affected subjects. The carrier had normal responses under all test conditions for dark-adapted, light-adapted, and rapid-flicker ERG stimuli. Figure 3Bshows the b-wave response function for the rod-isolating blue flash stimulus condition. Responses in two affected subjects (A2 and B1) were consistently below normal at all intensities. At the ISCEV (International Society for Clinical Electrophysiology of Vision) standard flash, the mean b-wave amplitude in normal subjects was 325 ± 83 μV (SD; n = 50), whereas two affected subjects had responses 2 to 3 SDs lower: subject A2 (168 μV, 1.9 μV SD lower) and subject B1 (72 μV, 3.0 SD lower). 

Rod Phototransduction.

As a consequence of the reduced rod b-wave in CNGB3-affected individuals, we critically assessed rod phototransduction parameters using the ERG. The dark-adapted maximum rod a-wave R _max amplitude determined from phototransduction modeling of subjects A1 (−239.8 μV) and A3 (−202 μV) was below normal mean (−380.82 ± 77.26 μV SD; range: −294.7 μV to −535.6 μV; n = 12). R _max in the other two affected subjects—A2, 346.7 μV; B1, 305 μV—were essentially normal and were within 1 SD of the normal mean. Rod R _max in control subjects was determined after computer subtraction of the cone component at each stimulus intensity. This was not necessary for the affected subjects because, in all cases, the cone component was diminished for even the brightest flash. 

Rod a-Wave Sensitivity.

Sensitivity was considered, first by normalizing the peak a-wave amplitude ^{16 20} at the brightest flash (4.77 log scot td-s) as shown in Figure 4Aand second, by fitting the leading edge of the a-wave with the model (Fig. 4B) . The normal mean was found by averaging the individual normalized a-waves of each normal subject. As shown in Figure 4A , the leading edge of the normalized a-wave of the affected subjects (normalized for the brightest flash response) was at the lowest end of the normal range. The photoresponse sensitivity (shown in Fig. 4Bas log of sensitivity from response modeling as a function of stimulus energy) shows that log S of the affected subjects was also at the lowest of normal across all intensities. Log S of subject B1 was within normal range at the brighter intensities from 12 individuals. 

Cone Responses.

All four affected subjects had nearly negligible photopic a- and b-waves for all stimulus intensities on a rod-suppressing background. We had recorded photopic ERGs on two of these subjects at younger ages with the same test equipment, and they had residual photopic b-wave responses at that time. Subject B1 had a 12-μV photopic b-wave when tested 6 years earlier (at age 51 years). Similarly, subject A1 was recorded 12 years before (at the age of 37) and had a 10-μV photopic b-wave. All this testing was performed on the same ERG equipment with the same flash intensity of 0.7 log cd-s/m². This provides evidence of progression of residual cone functional loss over a 6- to 12-year interval, even in mid-life. 

For carrier C1 (70 years of age), the cone phototransduction model was fitted to the leading edge of the cone a-wave (Fig. 5) . Cone R _max was 53.9 μV and within 2 SDs of control values (75.25 ± 16.3 SD; n = 12). Her cone response sensitivity log S was at the lower end of the normal range across all intensities, comparable to her affected daughters. Her age of 70 years may contribute to the reduction. Although this may reflect haploinsufficiency that impairs cone channel function, we are uncertain to what degree her age also contributed to the decrease in sensitivity. 

Discussion

The extended phenotyping of these two families with CNGB3 mutations showed several surprising features. First, affected individuals showed extensive macular RPE atrophy beyond any level previously reported for this condition. The cones retained some capacity for visual signaling into the adult years, as evidenced by residual photopic b-wave recording conditions and by minimal levels of color discrimination on testing with Ishihara color plates. This residual cone function, however, showed progressive loss in middle age by repeated photopic ERG testing in two patients in this study. Further, there was a clear manifestation of the carrier state in the heterozygous CNGB3 frameshift individuals at both young and older age. Carrier states are rarely reported for autosomal recessive retinopathies, even though they are well known in X-linked recessive retinal dystrophies. ^{27 28} Finally, although the CNGB3 protein subunit of the cyclic nucleotide-gated channel is known to be expressed only in cone photoreceptors, the rod-driven ERG response was abnormal at the level of both the a- and b-waves. 

Mutations in CNGB3 at the ACHM3 locus on chromosome 8q21 cause achromatopsia among the Pingelapese Islanders, by an S435F missense change that is common in that population. ^{5 6 29 30 31} The most commonly reported CNGB3 mutation in patients originating from Europe and the United States is a 1-bp deletion, c.1148delC, that accounts for approximately 70% of achromatopsia-associated CNGB3 alleles in this ethnicity. ^{32 33} The classic clinical features of achromatopsia include the absence of cone function, demonstrated by electroretinogram (ERG) testing, but with normal or nearly normal rod function. ^{3 6 32 34 35} The macular appearance in individuals with CNGB3 achromatopsia ranges from normal to mild granularity of the RPE ^{6 32 35} to macular atrophy and pigmentation. ³⁴  

Minimal abnormal pigment mottling extending into the midperipheral retina was described in one 47-year-old patient with CNGA3 mutations. ³² Fundus changes consistent with macular disease were noted in three unrelated individuals with CNGB3 mutations. ³⁴ However, the macular atrophy in our four affected individuals is far more extensive than was recognized in the previous literature. In a prior study of achromats with CNGB3 mutations, five individuals carrying the Thr383fs CNGB3 mutation in a heterozygous state were found to have a fundus appearance that was unremarkable across ages 20 to 77 years. ³⁵ Two of these, 47 and 77 years of age, were symptomatic and had subnormal acuity, mild cone dysfunction, and normal color vision; three subjects (20–74 years of age) were asymptomatic with no evidence of retinal dysfunction by visual field testing, OCT, ERGs, and fundus examinations ³⁵ This finding is in marked contrast to the RPE granularity that we observed in all three carriers (5, 9, and 70 years of age) in our family A with the Thr383fs mutant genotype. The early-stage macular RPE atrophy in the 70-year-old subject was readily captured by fluorescein angiogram. 

Ocular postmortem histology of human achromats showed disease in the retinal-foveal architecture, and the number and integrity of cone photoreceptors ranged from very few receptors, either rods or cones, near the macula, very few foveal cones, and a markedly reduced number of cones throughout the retina. ¹ Retinal histopathology of one achromatic subject showed a normal number of foveal cones but with abnormal shape and a highly reduced number of extrafoveal cones. ³⁶ A separate case showed the absence of foveal cones with a markedly reduced number of cones across the entire retina, and the cones near the fovea were abnormally broad. ³⁷ Summarizing, the available retinal anatomy of the monochromat shows that the number of cones was reduced when compared to normal, but mutation analysis was not performed in any of these individuals. Using OCT, Nishiguchi et al. ³⁵ found attenuation but not an absence of retinal layers, including the photoreceptor layer in the central fovea, in subjects homozygous for the common Thr383fs homozygous mutation in CNGB3. 

Affected male B1 has the same missense mutation Arg403Gln in one allele reported previously in compound heterozygous association with the common frameshift mutation Thr383fs and caused a progressive cone dystrophy in a three-generation consanguineous family rather than achromatopsia ³⁴ Direct comparisons are difficult, however, as our subject B1 also has an amino acid substitution Arg427Cys in CNGA3. It is not known whether the additional mutation detected in CNGA3 is adding to the phenotype of this patient. Despite the mutational differences, there is remarkable commonality between these two families. 

That subjects A1 and B1 demonstrated a progressive loss of cone function over the 6 to 12 years before current examination indicates that some residual cone function can remain at a younger age for the CNGB3 phenotypes described herein and that the condition is clinically progressive. Residual cone function (<2.0 μV) has been reported in 12 patients with achromatopsia with CNGB3 mutations using the full-field 30-Hz flicker photopic flicker ERG. ³⁵ Two canine CNGB3 mutation models of achromatopsia are the Alaskan Malamute and the German Shorthaired Pointer. ³⁸ Ophthalmoscopy shows normal fundi in adult dogs of both breeds. Cone ERGs are detectable in younger 3- to 6-week-old pups, but become nonrecordable in adult dogs. Adult dogs lack all cones, but the rods are functionally and structurally normal. ^{39 40}  

Progressive degeneration of cone photoreceptors has been shown in CNGA3 ^−/−-knockout mice, which have normal rod responses and no cone-mediated responses. ⁴¹ There was evidence of progressive loss of cones from the second postnatal week, with complete loss of ventral cones by the third month, whereas residual dorsal cones could be identified in 22-month-old CNGA3 ^−/− mice; rod morphology was intact. ⁴² This report resembles our findings in CNGB3-affected patients who had lost residual cone function over a 6- to 12-year interval. The progressive loss of cone function could be the result of a total loss of protein as seen in the CNGA3-knockout mice. ⁴² Truncating mutations such as a frameshift, in exons other than the last exon are likely to result in decay of the mRNA through nonsense mediated decay (NMD). This results in a total loss of CNGB3 protein. However, comparing CNGB3 patients directly with CNGA3 ^−/− mice is tenuous at best. In the case of frameshift mutations, the mechanism could be due either to the loss of protein caused by NMD or to the presence of abnormal protein if the truncated protein exists. 

In previous human studies, it has been reported that rod function in achromats is sometimes slightly subnormal. ^{6 35} ERG phototransduction modeling of rod function of the affected subjects in the present study remains technically normal for rod a-wave R _max amplitude and log S response sensitivity. However, values in all affected subjects are in the lower range of 12 normal control subjects including the scotopic b-wave, which in the context of the published histopathology could indicate a reduced number or function of rods. 

CNGB3-associated achromatopsia is one of the few diseases currently known to exhibit apparent rod dysfunction due to a defect in a cone photoreceptor specific gene. In the reverse case, cones frequently suffer a “bystander effect” and undergo progressive loss when rods are primarily affected in rod–cone dystrophy. In CNGB3 subjects A1 and A3, the b-wave was less impaired, whereas the a-wave was below the normal mean. Relative preservation of b-wave amplitude with a reduction in a-wave amplitude has been noted in the P23H rhodopsin transgenic rat. ^{43 44} Although the reason for this selective reduction in a-wave is not known, the proposed explanations include synaptic compensation or even “neuronal rewiring” after partial loss of rod photoreceptors and buffering by the large receptive fields of bipolar cells. ^{43 44} Indeed, ectopic synapses have been demonstrated between cone bipolar cells and rods in the absence of functional cones in the CNGA3 ^{− / −} mouse ⁴⁵ and between rod bipolar cells and cones in the degenerating retinas of RCS rat. ⁴⁶ In subjects A2 and B1, the opposite was true, and the a-wave was essentially normal, whereas the b-wave was impaired by two to three standard deviations below the normal mean amplitude, exhibiting a pattern of cone–rod dystrophy. Functional phenotyping of additional affected subjects may with CNGB3 mutations clarify the details further. 

 Supported by The Foundation Fighting Blindness, National Eye Institute Grants EY7003-CORE and R01-EY06094, Research to Prevent Blindness, NIH Intramural Research Program (PAS). Grants Wi1189/6-1 and 6-3 from the German Research Council (BW).

 Submitted for publication December 21, 2006; revised March 13, 2007; accepted May 21, 2007.

 Disclosure: N.W. Khan, None; B. Wissinger, None; S. Kohl, None; P.A. Sieving, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Genotypes and Mutations of CNGB3 in Two Families^*

Subjects	Age	Acuity	Fundus Appearance	Nucleotide Sequence Alteration	Protein Alteration	Allele Status
Family A			Heavy RPE granularity and pigmentary disruption in all homozygous subjects
Affected female A1	49	OD: 20/200	RPE thinning	c.1148delC	Thr383fs	Homozygous
		OS: 20/200
Affected female A2	45	OD: 20/200	Macular RPE mottling, RPE atrophy	c.1148delC	Thr383fs	Homozygous
		OS: 20/200
Affected female A3	40	OD: 20/200	Macular atrophy, RPE thinning	c.1148delC	Thr383fs	Homozygous
		OS: 20/400
Carriers
C1: Mother	70	OD: 20/40	Macular RPE mottling, RPE retraction	c.1148delC	Thr383fs	Heterozygous
		OS: 20/20
C2: Daughter of A1	9	OD: 20/30	RPE granularity	c.1148delC	Thr383fs	Heterozygous
		OS: 20/30
C3: Daughter of A3	5	OD: 20/20	RPE granularity	c.1148delC	Thr383fs	Heterozygous
		OS: 20/20
Family B
Affected male B1	57	OD: 20/300	Atrophic fovea, heavy RPE granularity	c.819-826del8	Pro273fs	Heterozygous
		OS: 20/300		c. 1208G→A	Arg403Gln
				CNGA3
				c.1279C→T	Arg427Cys	Heterozygous
Carriers
Father	84	Not available		c.819-826del8	Pro273fs	Heterozygous
				CNGA3
				c.1279C→T	Arg427Cys	Heterozygous
Mother	78	Not available		c.1208→A	Arg403Gln	Heterozygous

*  No mutations were detected in the CNGA3 gene in family A.

Figure 1.

 

Fundus appearance in subjects A1 (age 49), A3 (age 40), and B1 (age 57) showed minimal to extensive bilateral macular atrophy, extensive RPE granularity, and pigmentary disruption. Data are not shown for subject A2 (age 45).

Figure 2.

 

All three carriers showed RPE granularity. Subject C1 (age 70) manifested macular RPE mottling in the macula and inferior retina.

Table 2.

 

Ishihara Plates

Plate Number	Normal Response	Response of Person with Red-Green Deficiency	Response of Person with Severe Defect	SUB A1	SUB A2	SUB A3	SUB B1
1	12	12	12	12	12	12	12
2	8	3	x	26	x	x	x
3	29	70	x	x	x	7	x
4	5	2	x	5	5	5	5
5	3	5	x	3	3	3	3
6	15	17	x	15	17	13	15
7	74	21	x	74	71	71	74
8	6	x	x	x	x	x	x
9	45	x	x	x	x	x	x
10	5	x	x	5	6	2	x
11	7	x	x	7	7	5	7
12	16	x	x	26	16	x	x
13	73	x	x	x	x	71	x
14		5	x	x	x	x	x
15		45	x	x	x	x	x

 x, plate could not be read.

Table 3.

 

Sloan Achromatopsia Test

Test No./Color	1/Grey	2/Red	3/Yellow-Red	4/Yellow	5/Green	6/Purple-Blue	7/Red-Purple
Normal match	4.0	5.0	6.0	8.0	4.9	3.3	4.3
Our carrier	4.5	5.0	4.5	5.0	5.0	4.5	4.0
Mean achrom 26	4.0	3.5	4.8	6.0	5.6	4.7	3.9
A1	4.0	3.5	4.5	6.5	5.5	4.0	3.5
A2	4.25	3.5	4.5	6.5	5.75	4.5	3.5
A3	4.0	3.5	4.5	6.5	5.5	4.0	4.5
B1	5.0	3.5	5.5	7.5	5.5	4.5	4.0
Mean of 4 CNGB3 subjects	4.3	3.5	4.75	6.75	5.6	4.25	3.9

Figure 3.

 

(A) Representative ERG waveforms of a normal control subject, carrier C2, and affected subject A2. The ERG of carrier C2 was similar to the control, whereas the affected subject A2 showed subnormal rod responses and greatly diminished cone responses. (B) Dark-adapted b-wave amplitude ± SE as a function of flash intensity shows that affected subjects had b-wave amplitudes that were below normal mean. The lowest amplitudes were for subject B1 at all flash intensities.

Figure 4.

 

(A) Normalized rod a-wave at maximum flash intensity. Short-dashed black lines: normal range; long-dashed line: normal mean. Colored lines: subject responses. In all four affected subjects, the leading edge of the a-wave was on the slower side of normal. (B) Rod a-wave sensitivity of the four affected subjects determined from the model of activation of phototransduction. Log S in all four subjects was at the low end of normal range. Dashed line: control mean ± SE from 12 control subjects; shaded region: normal range.

Figure 5.

 

Cone a-wave sensitivity as a function of flash energy. Dashed line: normal mean ± SE of 12 control subjects; shaded region: the normal range. Carrier C1 had sensitivity at the lower end of normal but was still within normal range.

The authors thank Britta Baumann for excellent technical assistance, Jennifer Kemp for assistance with psychophysical testing, and Juanita Marner and Cathy Geer for assistance with the manuscript.