The loss-of-function mutation in
ABCA4 recently described in LR dogs
15 can cause clinical signs in the retina of affected dogs by1 year of age. The ophthalmoscopic signs are subtle and IR and FAF imaging facilitates detection of the abnormal appearance of the fovea-like area and surrounding area centralis. Although there are more or less scientific ways to convert the age of a dog to human years,
18,19 the onset is no doubt juvenile. The age of the youngest dog with evident ophthalmoscopic and cSLO findings in the fovea equivalent would approximately translate to a human subject in their teens or twenties depending on the age conversion formula preferred.
The localization and the focal FAF hypofluorescence in the fovea-like center of the area centralis mimic forms of childhood-onset STGD and correspond to the mildly abnormal foveal appearance and FAF patterns sometimes seen in young human patients.
10 Although a decrease in FAF may be caused both by loss of RPE and photobleaching of bisretinoid fluorophores in aging human patients,
20 we believe that the decrease in FAF in the fovea-like center and its surround in affected LRs is more likely to reflect loss of RPE rather than photobleaching of aged lipofuscin, as this was observed already in young dogs.
The canine fovea equivalent, surrounding area centralis and its horizontal extension, the visual streak, are all located in the tapetal fundus of the dog, where the RPE overlying the tapetum lucidum is non-pigmented.
16,21 Both the tapetal reflectivity and tapetal color change in puppies, but the tapetal reflection is considered to be adult-like within the first three months of life.
22 Hence, abnormal tapetal reflection in affected young dogs cannot be attributed to their youth. The marked decrease in overall tapetal reflection with age seen with short-wavelength cSLO (FAF-settings) but not evident on IR-images, has not been described previously to the best of our knowledge. We have not explored the reason for this decrease further, but it was observed regardless of genotype. It has been suggested that the number of tapetal cell layers decrease with age in the dog and thereby, both the tapetal reflectivity as well as the tapetal area decrease.
23 Reduced transmission of short-wavelength light through the aging optic media, neuroretina and RPE may also contribute to the fainter tapetal reflection.
RPE atrophy has been suggested both to precede and be secondary to photoreceptor degeneration in STGD1 patients.
24,25 Either way, the RPE may be overloaded with shedded outer segment material containing toxic bisretinoids when ABCA4 is dysfunctional in the photoreceptors. Additionally, functional ABCA4 in the RPE has also been shown to be important for maintaining both the RPE and photoreceptors.
3 In middle-aged affected LRs, the FAF-hyporeflection suggestive of RPE atrophy extended well outside the fovea-like area and into the area centralis and visual streak. In the oldest STGD dogs, the RPE atrophy had spread to the peripheral retina. This is in agreement with several reports on progressive RPE atrophy in both human patients and transgenic mice with mutations in the
ABCA4 gene.
26–28 RPE atrophy was not a typical finding on histopathology in our previous article,
15 but samples were taken more peripherally and nasally (opposite side to the fovea equivalent) and the results may therefore reflect either individual or regional differences or both. In old dogs, bleaching of the retinal fluorophores over the years may contribute to a reduction in FAF, as reported in human patients with some forms of recessive Stargardt disease.
20 We observed diffuse, increased FAF in the nontapetal area in old affected LRs. Lipofuscin has been described as the dominant fluorophore in the retina and was observed in the RPE of affected dogs in our previous study,
15 but other retinal fluorophores such as photooxidized bisretinoids also autofluoresce.
29 Thus accumulation of phototoxic bisretinoids (including A2E) is likely to contribute to the increase in FAF seen in
ABCA4InsC/InsC LRs.
The cSLO IR-hyporeflection, as well as the abnormal tapetal reflection and retinal vascular attenuation also seen on ophthalmoscopy was more evident in older affected dogs and not restricted to the area centralis surrounding the fovea equivalent indicating a diffuse, widespread retinal degeneration in old LRs. This is in agreement with electroretinographic, histopathologic and immunofluorescent findings in the old affected LRs previously reported.
15 Thus this loss-of-function mutation causes neuroretinal and RPE degeneration slowly spreading from the fovea-like area, into the midperiphery and further toward the peripheral fundus, mimicking the progression from Stargardt macular dystrophy to cone-rod dystrophy seen in some human patients with
ABCA4 mutations.
10,11,26,30–32 A more widespread retinal degeneration causing a reduction of both cones and rods was further supported by OCTs showing retinal thinning, most evident in middle-aged and older affected LRs, with normal appearing inner retina and thinning of the ONL. The thinning of the ONL in the fovea-like area and 0.5 mm nasally and temporally to this position was even statistically significant despite the low number of dogs included in this study. Because rods outnumber cones by far in both the area centralis and peripheral retina,
17 the ONL thinning indicates that not only cones have been lost, but also rods. The abnormal appearance of outer retinal structure in the fovea equivalent of young affected LRs mimic findings in childhood-onset STGD1.
10
Yellowish flecks are a hallmark in human STGD1 but may not be present in children with early-onset forms of the disease.
10 Furthermore, flecks are not typically seen in CRD associated with
ABCA4 mutations.
33–35 We were not able to convincingly detect flecks in or near the fovea-like area, either by ophthalmoscopy or cSLO. One reason may be that flecks are not a typical finding in LRs with this mutation; another potential reason is the inherent difficulty to detect such yellowish material in dogs where the tapetum lucidum often has a bright yellow to greenish reflection. None of the dogs reported in this current study developed the widespread mottled brownish deposits in the tapetal area that were present in one of the dogs in our previous report.
15
Interestingly, the middle-aged and older carriers for the mutation studied showed a mild phenotype characterized by accumulation of lipofuscin in the RPE in the fovea-like area. Although simple vision testing and ophthalmoscopy were considered unremarkable in these dogs regardless of age, both IR- and FAF-cSLO showed abnormal appearance in the fovea-like area (and very subtly in the visual streak). On OCTs, the ONL thickness in carriers fell in between that of age-matched, wildtype and affected LRs, although the difference was not statistically significant. The majority of human carriers for
ABCA4 mutations are not considered to show a clinically abnormal phenotype,
14 although heterozygotes for some mutations may have both abnormal retinal function and morphology causing visual impairment detectable in at least some patients on psychophysical testing.
12,13 Although very rare, a mild retinal phenotype has been reported in human heterozygotes for
ABCA4 mutations. A male with 20/20 visual acuity being heterozygous for the splice site variant c.5714+5G>A was reported to have parafoveal pisciform flecks corresponding to disruption of the EZ- and RPE-bands on OCT.
36 Furthermore, a fine granular FAF-pattern with peripheral punctate spots, a phenotype associated with human age-related macular degeneration patients, was recently attributed to monoallelic
ABCA4 mutations rather than risk increasing AMD alleles
37 again indicating that some ABCA4 mutations may cause a phenotype in carriers. The loss-of-function mutation in the LRs causes a premature translation stop codon, and our previous results indicate that the truncated mRNA-fragments are likely to be targeted by nonsense-mediated decay.
15 Such a serious mutation would therefore result in a loss of the ABCA4 protein, which is likely to be the reason for the abnormal morphology observed in elderly canine carriers.
The impact of the
ABCA4 loss-of-function mutation in Labrador retrievers, most of them field-trial type dogs, was variable, as reported by their owners and trainers. The performance of young, affected LRs ranged from normal to having unexpected problems detecting markings (falling birds or dummies) at a distance. Our attempts to crudely assess vision suggested that daylight vision was impaired in at least some dogs early in life, whereas concomitant impaired dim-light vision was seen mainly in old dogs. However, these old dogs, close to the end of a normal LR lifetime, still retained some vision allowing them to live a relatively normal life as companion animals, as reported by the owners. We were neither able to detect signs of visual impairment in heterozygotes for this mutation (some of them field-trial champions), nor did we receive anecdotal evidence from their owners suggesting that the performance of these dogs was affected even at old age. The lack of signs of impaired vision may also reflect that the methods used for testing vision were far too insensitive and that these dogs may rely less on high-acuity vision for solving tasks included in the daily life of a retriever. Furthermore, human carriers for
ABCA4 mutations have been reported to have normal visual acuity, although multifocal ERG revealed abnormal macular function.
12
We have previously reported on retinal function assessed with ERG in two LRs homozygous for and one LR heterozygous for the
ABCA4 loss-of-function mutation.
15 Further assessment of retinal function in both affected dogs and carriers would, of course, be valuable. However, we have been studying this mutation in privately-owned dogs, which limited both number and type of procedures the owners consent to. A colony of dogs with this
ABCA4 mutation will provide better opportunity to study this disease in greater detail.
In summary, the spontaneous, loss-of-function ABCA4 mutation results in abnormal appearance and morphology of the fovea-like center of the area centralis, including outer retinal thinning compared to age-matched wildtype LRs, abnormal segmentation of the outer retinal layers seen on OCT and focal loss of RPE, already in juvenile LRs. In older dogs, the abnormal appearance has spread into the area centralis and visual streak and later on, into the more peripheral fundus indicating a progression from a canine equivalent to a foveopathy to a diffuse cone-rod degeneration. Vision slowly deteriorates, but some vision seems to be retained throughout a normal LR lifetime. Older heterozygotes may show a mild phenotype with increased FAF in the fovea-like area, but visual impairment could neither be detected using simple testing nor was revealed in the history of such a dog.
Knock-out mouse models provided important information about
ABCA4 retinopathies but are not precise models of human disease.
38 Furthermore, the small size of the globe, the lack of a fovea or fovea-like area and difficulty to train mice for more advanced behavioral testing limit the usefulness of murine models. Because of similarities in retinal morphology between affected LRs and more severe forms of juvenile-onset STGD1 in human patients, as well as the human-like size of the globe in this species with a fovea-like area, we believe the affected LR is a suitable large-animal model for both basic science research and development of novel therapeutic interventions.