Introduction

Lipofuscin is derived from the shed and degraded photoreceptor outer segments and normally accumulates in the retinal pigment epithelium (RPE) with age [1–4]. An abnormal increase or depletion of lipofuscin manifests as high or low density areas respectively in fundus autofluorescence (AF) images [5] and such changes may aid in the detection and characterisation of a wide range of inherited retinal disorders, either by accentuating the visibility of fundus abnormalities or by revealing changes not accessible by routine examination or fluorescein angiography [5–13]. Lipofuscin has been shown to fragment when exposed to light [14] and its presence suggests continuing metabolic demand [5]. Absence of autofluorescence suggests either blocking of the incident radiation, photoreceptor cell death and RPE atrophy [5, 6] or disruption of the vitamin A cycle [15, 16].

Some patients with genetically different forms of retinitis pigmentosa manifest a ring of high density AF representing abnormal parafoveal accumulation of lipofuscin which usually encircles preserved foveal areas [17–20]. Similar parafoveal rings have recently been documented in other retinal dystrophies including some patients with Leber congenital amaurosis [21], Best disease [13], X-linked retinoschisis [22] and cone-rod dystrophy consequent upon mutation in GUCA1A [23], GUCY2D [24], RIMS1 [25, 26], or RPGR ORF15 [26, 27]. Abnormal annular AF has also been described in some cases of “cone dystrophy with supernormal rod ERG” consequent upon mutation in KCNV2 [28, 29]. Unlike RP, these disorders often result in atrophic macular changes, manifesting as low density AF within the ring.

The main aims of the current study were to review the electrophysiological phenotypes associated with annular AF abnormalities, and to examine two heterogeneous groups of patients in more detail by comparing indices of macular function associated with abnormal macular AF in rod-cone and cone-rod dystrophies.

Materials and methods

Autofluorescence imaging was performed using a scanning laser ophthalmoscope according to previously described techniques [5, 30]. Eighty one patients with abnormal AF of the posterior pole in the form of a high density parafoveal ring were reviewed. Sixty two had a clinical diagnosis of retinitis pigmentosa or Usher syndrome with a visual acuity of 6/9 or better and included 30 cases described previously [17]. Nineteen other individuals were ascertained from previous studies [21–23, 25–29] including 14 with cone-rod or cone dystrophy consequent upon mutation in RPGR ORF15 (4 cases), RIMS1 (8 cases) or GUCA1A (2 cases). Two subjects had “cone dystrophy with supernormal rod ERG” consequent upon mutation in KCNV2, two had RS1mutations and manifested rings in one or both eyes. One patient had Leber congenital amaurosis.

Full-field ERGs were performed according to extended testing protocols incorporating the ISCEV minimum standard [31] in order to assess generalised retinal function. A stimulus 0.6 log units greater than the ISCEV maximum was also used, to demonstrate better the a-wave under conditions of dark adaptation. Pattern ERGs evoked by high contrast checkerboard reversal were recorded according to ISCEV recommendations [32] using standard parameters; checkerboard size 12 × 15 degrees, check size 45′, Michelson contrast 0.98. The PERG P50 component was used as an index of macular function. Additional PERG testing was performed using a range of circular checkerboard fields ranging from 3 to 18 degrees in diameter presented in a random order [17]. Check size was constant at 45′. International-standard mfERGs [33, 34] and fine matrix mapping were performed in some cases. Fine matrix mapping measures rod and cone system sensitivity and has been described previously [18, 35, 36]. In brief, detection thresholds for a spot of light were determined at 1-degree intervals over selected 9 × 9-degree retinal areas. Data were presented both as sensitivity contours, illustrating the position and orientation of tested retinal locations and as three-dimensional threshold profiles, plotted using interpolated values at 0.25-degree intervals, obtained by Gaussian filtering.

Results

All patients were selected according to fundus autofluorescence that was characterised by an abnormal high density parafoveal ring (Figs. 1, 2, 4–8). Sixty two with a clinical diagnosis of retinitis pigmentosa or Usher syndrome and a visual acuity of 6/9 or better had evidence of preserved RPE within the ring. Nineteen others had either cone or cone-rod dystrophy, “cone-dystrophy with supernormal rod ERG”, X-linked retinoschisis or Leber congenital amaurosis. Many of the older non-RP cases had evidence of macular RPE atrophy within the ring (Figs. 2b–d, 7c, 8b–c). Conversely, AF imaging in some RP patients showed mild to moderate atrophic RPE changes within the vascular arcades but external to the ring (Figs. 4d and 5e).

Fig. 1
figure 1

Full-field ERGs, PERGs and AF in 3 patients with rod-cone dystrophy (a–c) including a patient with RP18 (a) and Usher syndrome (b). Row D shows full-field ERGs and AF in a case of Leber congenital amaurosis; ERGs and the AF image were obtained in the presence of nystagmus and are consequently noisy. Normal examples are shown for comparison (e). LU indicates log units greater (+) or less (−) than the ISCEV standard flash

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Fig. 2
figure 2

Full-field ERGs, PERGs and AF in cone rod-dystrophy consequent upon mutation in RPGR (a), RIMS1 (b), “cone dystrophy with supernormal rod ERG” (c) and in a case of X-linked retinoschisis (RS1; d). Normal examples are shown for comparison (e). LU indicates log units greater (+) or less (−) than the ISCEV standard flash

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Figures 1 and 2 show representative full-field ERGs and PERGs in eight individuals with different retinal dystrophies. Figure 1 shows data from 3 subjects with rod-cone dystrophy and normal visual acuity including a case of autosomal dominant RP18 (Fig. 1a) and Usher syndrome (Fig. 1b). Figure 1d shows undetectable ERGs in a patient with Leber congenital amaurosis; ERGs are contaminated by the effects of nystagmus but no ERG is detectable, in keeping with severe generalised photoreceptor dysfunction. Figure 2 shows representative examples of cone-rod dystrophy consequent upon mutations in RPGR (Fig. 2a), RIMS1 (Fig. 2b) and KCNV2 (“cone dystrophy with supernormal rod ERG”; Fig. 2c). Figure 2d shows the ERG and AF findings in a patient with X-linked retinoschisis.

In the 62 RP cases PERG P50 components varied between normal (>2 uV) and undetectable, consistent with varying degrees of macular involvement. There was no significant correlation with the bright flash ERG a-wave amplitude or 30 Hz flicker ERG amplitude. Figure 3 shows high positive correlation between PERG P50 and mean ring radius (r = 0.83, P < 0.0005). Data are shown from one RP patient that was tested comprehensively (Fig. 4); PERGs were normal to the smallest diameter checkerboard but minimal enlargement was seen as the stimulus field size was increased (Fig. 4b). Multifocal ERGs showed widespread reduction with relative preservation of the response associated with the central stimulus element (Fig. 4a), consistent with a central island of visual field preservation (Fig. 4c). The internal edge of visual field constriction corresponded closely with the ring of high density, as shown by photopic FMM (Fig. 4d). Scotopic fine matrix mapping revealed rod sensitivity losses that were severe and that encroached upon the central macula within the ring. Additional examples of mfERGs, visual fields and fine matrix mapping in RP patients are shown in Figs. 5 and 6.

Fig. 3
figure 3

Comparison of mean ring radius with PERG P50 in 62 patients with rod-cone dystrophy (RP; broken linear regression line) and normal visual acuity and in 19 patients with other retinal dystrophies, including 4 cone or cone-rod dystrophy cases in which there was a detectable PERG (solid regression line)

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Fig. 4
figure 4

Multifocal ERGs (a), small field PERGs (b), Humphrey visual field (c) and photopic (d) and scotopic (e) fine matrix mapping in a patient with a clinical diagnosis of RP. Diamonds and error bars in (b) show mean values and standard deviations for 8 normal subjects; triangles and squares show patient data from right and left eyes. Contour plots (d and e) show sensitivity gradients over tested retinal locations; corresponding 3-D plots show retinal location (abscissa, degrees) and thresholds (ordinate, log units). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Normal photopic and scotopic values are plotted in Fig. 5a and d

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Fig. 5
figure 5

Contour sensitivity plots (rows 1 and 3) and 3-D threshold plots (rows 2 and 4) obtained in representative normal subjects (a, d) and in 3 RP patients (b, e and c, f). Subjects were tested under photopic (a–c) and/or scotopic conditions (d–f). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units). Corresponding photopic FMM in individual (e) has been published elsewhere [18]. Normal 3-D plots show averaged data from 14 (a) or 12 (d) normal subjects

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Fig. 6
figure 6

AF images, mfERGs and corresponding Humphrey visual fields in 3 patients with RP and normal visual acuity

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Pattern ERGs were detectable in only 4 patients with cone or cone-rod dystrophy (GUCA1A, RPGR or RIMS1, Fig. 3). Pattern ERG P50 was inversely related to ring size in these subjects (Fig. 3). Data from one subject with cone-rod dystrophy (RIMS1) are shown in Fig. 7. Multifocal ERG showed widespread reduction with only relative preservation of the central response (Fig. 7a). Standard Humphrey visual fields showed a central scotoma and some superior field loss (Fig. 7b). Fine matrix mapping revealed severe threshold elevation across the macula but with relatively preserved photopic sensitivity over a central island of preserved RPE AF (Fig. 7c). Threshold values inside the ring are maximally elevated over a concentric atrophic area and show a gradient of increasing sensitivity over the arc of high density (Fig. 7c and e). The PERGs in this patient were undetectable (data not shown). Additional examples of fine matrix mapping in RIMS1 and RPGR patients are illustrated in Fig. 8. Central RPE atrophy was not always present (Figs. 2a, 8a).

Fig. 7
figure 7

Multifocal ERGs (a), Humphrey visual field (b) and photopic (c, d) and scotopic (e, f) fine matrix mapping in a patient with cone-rod dystrophy consequent upon RIMS1 mutation. Labelling (x) shows correspondence between the orientation of contour and 3-D threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units). Threshold values for half the tested area have been removed from c and e, to expose the foveal values that would otherwise be obscured

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Fig. 8
figure 8

Contour sensitivity plots (rows 1 and 3) and 3-D threshold plots (rows 2 and 4) obtained in 3 patients with cone-rod dystrophy consequent upon RPGR (column 1) or RIMS1 mutations (columns 2 and 3). Subjects were tested under photopic (a–c) and scotopic conditions (d–f). Labelling (x) shows correspondence between the orientation of contour and threshold plots. Abscissa shows retinal location (degrees), ordinate axes show threshold (log units)

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Discussion

This study reviews a heterogeneous group of 81 patients with genetically-determined retinal diseases that manifest a common feature on fundus autofluorescence imaging in the form of a parafoveal ring of high density. Patients with RP and normal visual acuity had rings that encircled preserved central AF. Mild to moderate atrophic changes were occasionally detected within the vascular arcades but eccentric to the ring (Figs. 4d, 5e). In the other retinal dystrophies that were examined in this and in previous studies [25–27], the ring could also encircle preserved central AF but in older subjects there was often central RPE atrophy within the annulus and preserved AF at more eccentric locations.

As patients with RP or different forms of retinal dystrophy can have indistinguishable AF abnormalities, the AF appearance cannot be used to establish a diagnosis in such cases. Non-specific annular increases in AF may be associated with a wide variety of distinctive or pathognomonic full-field ERG changes that are essential for accurate diagnosis and functional phenotyping (see below).

The data from 62 patients with RP and normal visual acuity show a high positive correlation between ring size and the PERG P50 component extending and confirming findings in a cohort of 30 of these cases [17], demonstrating the robust nature of this relationship. The findings are corroborated by high spatial resolution fine matrix mapping, visual field data and mfERG testing that shows preserved photopic sensitivity within central areas bordered by the ring, consistent with previous reports [18–20, 37]. Scotopic sensitivity losses encroach upon central macular areas suggesting that rod-system dysfunction precedes abnormal parafoveal accumulation of lipofuscin and progressive visual field loss [18]. Serial data indicate that the rings may vary greatly in terms of their stability; to date only 3 cases have been reported in which progressive ring constriction has occurred [37]. The rate of AF ring constriction may prove to be of prognostic value in predicting retention of visual acuity and visual field preservation, but further monitoring is required.

In addition to RP, the parafoveal ring of high density may occur in cone or cone-rod dystrophy consequent upon mutation in GUCA1A [23], GUCY2D [24], RPGR [25, 26], RIMS1 [26, 27], in “cone dystrophy with supernormal rod ERG” (KCNV2) [28], in X-linked retinoschisis (RS1) [22] and in Leber congenital amaurosis [21]. Similar AF findings have also been documented in Best Disease [13] and other maculopathies [10]. In young patients with cone-rod dystrophy, small rings may have preserved central AF and are similar to those seen in RP cases [26]. Older individuals tend to manifest central atrophic changes that are encircled by the ring and there may be a central island of RPE preservation. Fine matrix mapping suggests that the abnormal accumulation of lipofuscin in cases of cone-rod dystrophy represents a transitional stage between relatively preserved parafoveal function and severe central dysfunction that is likely to precede central atrophy [26, 27]. In patients with cone-rod dystrophy consequent upon RPGR or RIMS1 mutations, serial studies have recently demonstrated ring expansion [26], suggesting an expanding front of macular photoreceptor dysfunction. This contrasts with RP where the opposite occurs [37]. It is possible that rings associated with other causes of maculopathy may also expand with time as lesions become larger with age. It is noted that RPGR mutations are more commonly associated with X-linked retinitis pigmentosa [38] with visual acuity reduction [39]; in the current study not all patients underwent genetic screening but none were known to have X-linked RP.

Lipofuscin accumulation in the RPE is likely to reflect metabolic activity which is largely determined by outer segment renewal. There is evidence of light-induced degradation of lipofuscin [14], so that its presence would depend upon continuing metabolic demand. Evidence from RCS rats indicates that failure of RPE cells to phagocytose outer segments results in reduced lipofuscin levels [40]. Lipofuscin formation is almost completely dependent on a normal visual cycle and on the availability of dietary vitamin A [41]. In the RPE65 knockout mouse, the visual cycle is impaired and fluorescence is reduced [42]. Similarly, patients with RPE65 mutations can have reduced autofluorescence in the presence of near-normal fundi and OCT findings that do not suggest reduction in the photoreceptor layer thickness [15, 16]. It is widely accepted that photoreceptor degeneration results in atrophy and reduced rather than stable levels of autofluorescence and it is likely that autofluorescence is lost within a few weeks of photoreceptor loss. The presence of foveal or parafoveal AF in the photoreceptor dystrophies outlined in the current study, suggest that photoreceptors are likely to be intact and thus may be amenable to functional rescue. Recent developments, for example in the field of gene therapy, make early identification of candidate patients increasingly important.

Patients with RP classically present with impaired night vision and visual field constriction, consistent with generalised retinal dysfunction involving rod more than cone photoreceptors. In the early stages the fundi may be near normal and rod-cone dystrophy is established by full-field ERG testing (Fig. 1). In patients with normal visual acuity, the degree of macular sparing cannot be predicted from the severity of peripheral dysfunction. The cone and cone-rod dystrophies are typically characterised by progressive worsening of visual acuity, dyschromatopsia, and photophobia with eventual central scotomata and peripheral field abnormalities. Ophthalmoscopic abnormalities, when present, are generally confined to the macula. Photopic full-field ERGs are typically delayed and subnormal with milder scotopic ERG abnormalities (Fig. 2a and b). A notable exception is the autosomal dominant cone dystrophy consequent upon mutation in GUCA1A; cone-mediated ERGs show amplitude reduction without significant implicit time delay [23] and although not diagnostic, may enable focussed mutational screening. Patients with “cone dystrophy with supernormal rod ERG” exhibit a wide range of fundus AF abnormalities including ring-like or bull’s eye changes, central atrophy or increased foveal AF [28]. Full field ERGs are pathognomonic in this disorder (Fig. 2c), have recently been shown to be consequent upon mutation in KCNV2 and have been described in detail [28, 29]. X-linked retinoschisis is usually associated with an electronegative bright flash ERG in keeping with inner retinal dysfunction (Fig. 2d); two cases (aged 47 years and 49 years) with annular AF abnormalities have been documented within a series of seven atypical but genetically confirmed cases [22]. The rings of high density in these individuals surround areas of central atrophy and may represent an outer boundary of macular photoreceptor dysfunction in addition to generalised inner rather than outer retinal disease. Commonly, younger individuals with X-linked retinoschisis manifest stellate macular lesions that are also visible in AF images [43].

Conclusions

A parafoveal ring of high density autofluorescence is a non-specific manifestation seen in different retinal dystrophies. Electrophysiology remains essential for accurate diagnosis. The high correlation of autofluorescence with PERG, mfERG and FMM demonstrates that AF abnormalities have functional significance and may be an important parameter in the monitoring of these patients. Autofluorescence may be of prognostic value and may help identify suitable patients and retinal areas amenable to future therapeutic intervention.