Human Retinal Disease from AIPL1 Gene Mutations: Foveal Cone Loss with Minimal Macular Photoreceptors and Rod Function Remaining

Abstract

Purpose.: To determine the human retinal phenotype caused by mutations in the gene encoding AIPL1 (Aryl hydrocarbon receptor-interacting protein-like 1) now that there are proof-of-concept results for gene therapy success in Aipl1-deficient mice.

Methods.: Leber congenital amaurosis (LCA) patients (n = 10) and one patient with a later-onset retinal degeneration (RD) and AIPL1 mutations were studied by ocular examination, retinal imaging, perimetry, full-field sensitivity testing, and pupillometry.

Results.: The LCA patients had severe visual acuity loss early in life, nondetectable electroretinograms (ERGs), and little or no detectable visual fields. Hallmarks of retinal degeneration were present in a wide region, including the macula and midperiphery; there was some apparent peripheral retinal sparing. Cross-sectional imaging showed foveal cone photoreceptor loss with a ring of minimally preserved paracentral photoreceptor nuclear layer. Features of retinal remodeling were present eccentric to the region of detectable photoreceptors. Full-field sensitivity was reduced by at least 2 log units, and chromatic stimuli, by psychophysics and pupillometry, revealed retained but impaired rod function. The RD patient, examined serially over two decades (ages, 45–67 years), retained an ERG in the fifth decade of life with abnormal rod and cone signals; and there was progressive loss of central and peripheral function.

Conclusions.: AIPL1-LCA, unlike some other forms of LCA with equally severe visual disturbance, shows profound loss of foveal as well as extrafoveal photoreceptors. The more unusual late-onset and slower form of AIPL1 disease may be better suited to gene augmentation therapy and is worthy of detection and further study.

There are currently 15 known molecular forms of Leber congenital amaurosis (LCA), and many animal models of human LCA are available for proof-of-concept studies. ^1,2 Gene therapy is efficacious in animal models of six of the LCA molecular subtypes: RPE65, ^3,4 MERTK, ⁵ LRAT, ⁶ RPGRIP1, ⁷ GUCY2D, ^8,9 and AIPL1. ^10,11 Translation from the laboratory to human clinical trials has occurred to date in only the RPE65 form of LCA. ^{12 –16} A key question that was asked in RPE65-LCA patients before the onset of human trials was whether the detailed phenotype of the human disease was similar enough to that in the animal models to justify a clinical trial. ^4,17  

The recent success of gene therapy in Aipl1-deficient mice raises the question of whether human AIPL1 disease is amenable to treatment. Human gene replacement therapy would seem feasible in this recessive photoreceptor disease caused by mutations in a relatively small gene that has already been carried into photoreceptors by several adenoassociated viral pseudotypes. ^10,11 What do we know to date of the retinal phenotype of AIPL1-LCA patients? There are brief clinical descriptions that have accompanied the reports of molecular identification ^18,19 ; clinical evaluations of AIPL1 patients, specifically or with other genotypes ^{20 –23} ; and donor retinal tissue studies. ^24,25 Frequently described findings include very low visual acuity even at young ages; no measurable visual fields; hyperopia; nondetectable electroretinograms; and pigmentary retinopathy with prominent maculopathy. An optical coherence tomography scan in a 22-year-old AIPL1-LCA patient showed abnormal laminar architecture. ²²  

Given that gene therapy for human AIPL1 retinal disease may be possible, based on the recent murine proof-of-concept results, ^10,11 we studied in detail the retinal structure and function of a molecularly clarified group of these patients, most of whom had the clinical diagnosis of LCA and one with later-onset retinal degeneration. 

Methods

Subjects

Eleven patients with retinal degeneration and AIPL1 mutations (Table 1, some with more than one examination) were included. They had a complete eye examination, including kinetic and static perimetry and electroretinography. ^26,27 Institutional review board approval and informed consent were obtained before the study, and the procedures adhered to the Declaration of Helsinki. 

Table 1.

 

Clinical and Molecular Characteristics of the AIPL1 Patients

Family, Patient	Age at Visit (y)/Sex	AIPL1 Allele 1	AIPL1 Allele 2	Visual Acuity*	Refractive Error (Spherical Equivalent)*	Kinetic Visual Field Extent (V-4e)†	Keratoconus*	Cataract*
Leber Congenital Amaurosis
Family 1
P1	1/M	p.Trp278X	p.IVS2–2A>G	FL	+5.00	UP	N	N
P2	5/M	p.Trp278X	p.IVS2–2A>G	≤1/200	+6.00	UP	N	N
Family 2
P1	0.5/F	p.Trp278X	p.Val33fs	FL	+6.00	UP	N	N
	7			HM	+5.00	≤1	N	N
	16			HM	+2.00	≤1	N	N
Family 3
P1	3/M	p.Val71Phe	p.Trp72X (c.216G>A)‡	CF-2ft	NA	UP	N	N
	8			HM	NA	ND	N	N
Family 4
P1	23/F	p.Cys89Arg	p.Trp72Arg	LP	+4.00	ND	Y	Y
Family 5
P1	27/M	p.Val71Phe	p.Val71Phe	HM / 2/200	−0.50	ND	N	N
Family 6
P1	33/M	p.Trp278X	p.Gly262Ser	CF-2ft	−5.00	≤1	N	Y
P2	40/F	p.Trp278X	p.Gly262Ser	HM	+4.50	NP	N	Y
Family 7
P1	39/M	p.Trp278X	p.Leu241del	LP	+5.00	≤1	N	N
P2	40/F	p.Trp278X	p.Leu241del	LP	+8.00	ND	N	Y
Later-Onset Retinal Degeneration
Family 8
P1	45/F	p.Trp278X	p.Gly122Arg (c.364G>C)‡	20/60 / 20/400	+2.00	16	N	N/N
	49			20/100 / 20/400	+2.00	12.2	N	N/Y
	55			20/100 / 1/200	+2.50	13	N	Y/Y
	60			20/400 / HM	+2.50	10.9	N	Y/Y
	62			20/400 / HM	+1.50	6.2	N	Y/Y
	67			20/400 / HM	+2.50/+1.00	0.95/ND	N	Y/P

 ND, not detectable; NA, not available; N, no; Y, Yes; UP, unable to be performed; FL, follows light; CF, count fingers; HM, hand motion; LP, light perception; P, pseudophakia.

*  Similar in the two eyes; otherwise, specified individually, as RE/LE.

†  Expressed as a percentage of normal mean for V-4e; 2 SD below mean equals 90%; average both eyes; similar in the two eyes unless specified as RE/LE.

‡  Novel mutation.

Visual Function

Full-field Sensitivity.

Absolute sensitivity of visual perception of flashed lights was determined by using full-field stimuli. Techniques, methods of data analysis, and normal results have been described. ^28,29 Dark-adapted sensitivity to the full-field stimulus test (FST) was measured with white, blue, and red, 200-ms flashes using an LED based, computer-driven stimulator (Colordome; Diagnosys LLC, Littleton. MA). Sensitivity loss was defined as the difference between mean normal and patient sensitivities to white stimuli. The sensitivity difference (blue minus red) in responses to chromatic stimuli was used to assess whether rods, cones, or both photoreceptor systems mediated perception. Cone-mediated perception is expected to yield a much smaller sensitivity difference (blue minus red, ≤3.1 dB) than rod-mediated perception (blue minus red, ≥19.3 dB). Mixed mediation refers to difference values between those limits and indicates that rods mediate blue detection and cones mediate red detection. ²⁹  

Pupillometry.

The direct transient pupillary light reflex (TPLR) was elicited and recorded in AIPL1-LCA patients (n = 7) and normal subjects (n = 13) according to a published methodology. ^16,30 TPLR luminance response functions were derived from responses to increasing intensities of short-duration (0.1 second) green (500 nm, from −6.6 to +2.3 log scot-cd · m⁻²) and orange (600 nm, from −4.5 to +1.3 log scot-cd · m⁻²) stimuli, presented monocularly in the dark-adapted state. The pupil images were digitized by two instruments simultaneously. A digital image processor (RK-706PCI, ver. 3.55; Iscan, Inc., Woburn, MA) sampled the horizontal pupil diameter at 60 Hz and a video digitizer (PIXCI SV4 board, ver. 2.1; Epix, Buffalo Grove, IL) produced a computer file of the video sequence. Each recording epoch lasted for 5.7 seconds, with a 1-second prestimulus baseline. Manual measurements of the digitized video sequence were performed when automatic measures were complicated by nystagmus or blink artifacts. TPLR amplitude was defined as the difference between the pupil diameter at a fixed time (0.9 seconds) after the onset of the stimulus and the prestimulus baseline. TPLR response threshold was defined as the stimulus luminance that evoked a criterion (0.3 mm, limit of spontaneous oscillations in pupil diameter; ³⁰ ) amplitude. LCA patients were expected to have an insensitive TPLR ³⁰ ; photoreceptor mediation was thus explored using the brightest pair of scotopically matched (1.3 log scot-cd · m⁻²) orange and green stimuli, so that responses would be available in most patients. This pair of stimuli produced nearly identical amplitudes in normal subjects. Mean (±2SD) differences (green minus orange) in TPLR amplitude responses to these stimuli in normal subjects defined a range (+0.15 to −0.28 mm) wherein rod photoreceptor mediation of the TPLR could be expected. 

En Face Imaging

En face imaging was performed with a confocal scanning laser ophthalmoscope (HRA2; Heidelberg Engineering GmbH, Heidelberg, Germany). Retinal and subretinal features were imaged with 820 nm near-infrared (NIR) light in the reflectance (REF) mode. ³¹ The health of the retinal pigment epithelium (RPE) was estimated with recently developed reduced-illuminance autofluorescence imaging (RAFI) methods. Unlike conventional autofluorescence imaging, RAFI minimizes the absorption of imaging light by rod and cone opsins and lipofuscin and thus addresses the possibility of accelerating the natural history of the disease. Two types of RAFI were used. ^{32 –36} NIR-RAFI allowed detection of fluorescence emissions dominated by the melanin by using 790-nm excitation light and a band-pass (805–900-nm) blocking filter. SW-RAFI on the other hand, mapped the distribution of emissions normally dominated by lipofuscin by using a 488-nm excitation light and a band-pass (500–750-nm) blocking filter. RAFI images were obtained with a sensitivity setting of 95%. NIR-REF, NIR-RAFI, and SW-RAFI modalities were performed in the high-speed mode; either a 30° × 30° square region or a 55° diameter circular region was sampled onto a 768 × 768 pixel image, and video segments up to 10 seconds in length were obtained at an acquisition rate of 8.8 Hz. The automatic real-time (ART) averaging feature of the manufacturer's software was used whenever possible. When ART failed, the images were exported from the manufacturer's software and analyzed as previously described. ^{32 –36} Neighboring regions were digitally stitched by manually specifying corresponding retinal landmark pairs. 

Optical Coherence Tomography

Retinal cross sections were obtained with Fourier-domain (FD) optical coherence tomography (OCT) imaging (RTVue-100; Optovue Inc., Fremont, CA). Our recording and analysis techniques have been published. ^{35,37 –40} For this work, the “Line” protocol of the FD-OCT system, with greater speed of acquisition, was used to obtain 4.5- or 9-mm-long scans composed of 1019 longitudinal reflectivity profiles (LRPs) acquired in ∼4 ms. Overlapping, nonaveraged, OCT scans were used to produce a digital montage covering up to 9-mm eccentricity from the fovea along the horizontal and vertical meridians. All scans used for the montage included the foveal depression, to ensure unambiguous retinal localization. Further, a video fundus image was acquired immediately after each OCT scan. Three-dimensional FD-OCT raster scans (“3-D Macula” scans with 101 lines of 513 LRPs each or “Raster” scans with 17 lines of 1020 LRPs, each covering 6 × 6 mm) were performed for topographic analysis. In one patient (F3,P1), 3-D volume scans (25 lines covering 6 × 4.5 mm) were available from a different FD-OCT instrument (Spectralis HRA+OCT; Heidelberg Engineering, Heidelberg, Germany). 

Postacquisition processing of OCT data was performed with custom programs (MatLab 6.5; MathWorks, Natick, MA). LRPs making up the OCT scans were aligned by straightening the major RPE reflection. The outer photoreceptor nuclear layer (ONL) was defined in regions of scans showing two parallel stereotypical hyporeflective layers sandwiched between the RPE and vitreoretinal interface. Inner retinal thickness was defined as the distance between the signal transition at the vitreoretinal interface and the sclerad boundary of the inner nuclear layer (INL) or the single hyporeflective layer continuous with the INL. In normal subjects, the signal corresponding to the RPE was assumed to be the most sclerad peak within the multipeaked, scattering-signal complex, deep in the retina. In abnormal retinas, the presumed RPE peak was sometimes the only signal peak deep in the retina, but there could also be other major peaks. The RPE peak was specified manually by considering the properties of the backscattering signal originating from layers vitread and sclerad to it. In the case of the one patient recorded with the Spectralis instrument, LRP data were not available, and ONL, inner retinal, and total retinal thicknesses were measured directly from grayscale images. For topographic analysis using the RTVue instrument data, en face images of integrated backscatter intensity were generated from each group of “3D Macular” scans. Lines corresponding to eye movement were discarded using the continuity of blood vessel patterns on the generated en face images. The location and orientation of remaining lines were determined relative to retinal features (blood vessels, optic nerve head) and LRPs were allotted to regularly spaced bins (0.3 × 0.3 mm²) in a rectangular coordinate system centered at the fovea; the waveforms in each bin were aligned and averaged. ONL and inner retinal thickness were measured as just described. Missing data were interpolated bilinearly, thicknesses were mapped to a pseudocolor scale, and fundus landmarks were overlaid for reference. 

Results

Molecular and Clinical Features of AIPL1-Associated Disease

Ten of the 11 patients were diagnosed as LCA (Table 1). These patients had severe visual disturbances in infancy or early childhood, nystagmus, and nondetectable electroretinograms (ERGs). All but two patients had hyperopia. One patient (F4,P1) had keratoconus; four patients (ages 23–40 at the time of examination) showed posterior subcapsular cataract (Table 1). F8,P1 had a later-onset retinal degeneration. 

All patients were compound heterozygous for mutations in the AIPL1 gene with the exception of one homozygote. The W278X AIPL1 mutant allele was shared by eight of the patients, representing five families. Other AIPL1 alleles included another premature stop mutation, missense mutations, insertions, deletions, and an intron splice site mutation. Most of these alleles have been reported to be associated with LCA, ^19,41 but two variants are novel (Table 1). Ancestry in 8 of 11 patients was northern European (German, Austrian, or Dutch) or British Isles on both maternal and paternal sides. F3,P1 had Swedish ancestry on the paternal side and North African Jewish ancestry on the maternal side. F4,P1 had northern European ancestry on the paternal side and Puerto Rican ancestry on the maternal side. F5,P1 was of North African Jewish descent on both sides. 

En Face Imaging Shows Macular Abnormalities and Regions of Far Peripheral Preservation

The retina-wide appearance of AIPL1-LCA was evaluated in wide-field NIR-REF images across a retinal extent of 40 mm (130°) in F6,P1 and compared with images of the same area in a representative normal subject (Fig. 1A). The AIPL1-LCA patient at age 33 showed a peripapillary region of choroidal visibility. Starting beyond the eccentricity of the vascular arcades was a wide midperipheral annulus with bone-spicule pigmentation. Peripheral to this annulus (Fig. 1A, arrowheads) there was an abrupt transition to homogeneous NIR-REF appearance suggestive of relatively preserved RPE health in the far periphery. Wide-field NIR-REF imaging results available in five other patients (F2,P1; F4,P1; F6,P2; F7,P1; and F7,P2, data not shown) were comparable to that of F6,P1. 

Figure 1.

 

En face imaging in patients with AIPL1-LCA. (A) Digitally stitched wide-field, near-infrared, reflectance (NIR-REF) imaging in F6,P1 compared to a representative normal subject. Arrowheads: far peripheral transitions from bone-spicule pigment to relative homogeneity. (B) Central regions of F2,P1 (left) and F7,P2 (right). Melanin abnormalities are visible on reduced-illuminance autofluorescence imaging (RAFI) with NIR light and lipofuscin abnormalities are demonstrated on RAFI with short-wavelength (SW) light. Insets: representative normal images for both modalities. Images are individually contrast stretched for visibility of features and shown as right eyes for comparability. Bar, 5 mm.

RPE health within the macular region and extending into perimacular areas was further evaluated to study lesions at different stages of disease (Fig. 1B). In F2,P1 at age 16, NIR-REF imaging showed a mottled appearance suggesting RPE pigmentation abnormalities. NIR-RAFI, which is normally (Fig. 1B, inset) dominated by melanin fluorophores, showed a bright spot at the fovea of F2,P1 encircled by a parafoveal annular region demonstrating lower signal. On SW-RAFI, which is normally dominated by lipofuscin fluorophores, there was a relatively reduced signal in the parafoveal region of F2,P1 surrounding a local hyperautofluorescence corresponding to the anatomic fovea (Fig. 1B). The perifoveal region extending to vascular arcades showed definite fluorescence on NIR- and SW-RAFI except for the optic nerve and the major vasculature. Uniformly homogeneous appearance of the SW-RAFI taken together with the lack of a choroidal pattern on NIR-RAFI supports the existence of a continuous layer of melanized RPE cells across most of the macula. The relative hyperautofluorescence in the perifoveal region would normally be interpreted as better preserved RPE. Alternatively, however, this region may represent a stressed RPE demonstrating hyperautofluorescence due to oxidation of its melanin and lipofuscin fluorophores. ^42,43 Counterintuitively, the parafoveal ring of relative hypoautofluorescence may represent the healthiest RPE in this retina. 

F7,P2 at age 40 (Fig. 1B, right column) showed a large oval central macular region of hyperreflection with visible choroidal vessels on NIR-REF imaging. A choroidal pattern was also visible on NIR-RAFI. Taken together with the lack of signal on SW-RAFI, it is likely that this region corresponds to complete atrophy of the RPE. In the region surrounding the central atrophy, there was lower NIR-REF signal, a thin ring of NIR-RAFI hyperautofluorescence and a wider region of relatively homogeneous RPE melanin and lipofuscin signals on NIR-RAFI and SW-RAFI, respectively. All en face imaging findings were consistent with an annular region of retained RPE health surrounding the atrophy. Of note was the peripapillary loss of RPE and the transition to severe RPE atrophy in the midperipheral region. 

Cross-Sectional Retinal Imaging: Foveal Photoreceptor Losses

Retinal laminar architecture by high-resolution, cross-sectional OCT imaging in seven AIPL1-LCA patients, ranging in age from 9 to 40 years, was compared with that of normal subjects (Fig. 2). The central 15-mm scan of retina along the vertical meridian in a normal 23-year-old subject (Fig. 2A) illustrates the foveal depression and the hypo- and hyperreflective layers that have been shown to have a predictable relationship to histologically defined layers. ^37,38 Lamination in the six patient scans was abnormal (Fig 2A). In all patients, a foveal depression was identifiable; foveal retinal thickness measurements of these AIPL1-LCA patients ranged from 40 to 120 μm, which is significantly reduced (P < 0.001) below normal (n = 27; ages, 5–58; mean ± 2SD, 207 ± 35.6 μm). In the patients, the foveal ONL was either barely discernible or not detectable, and inner segment and outer segment laminae were also not visible in the fovea. Parafoveal ONL, however, was barely detectable in some of the patients (highlighted on scans in Fig. 2A). 

Figure 2.

 

Retinal laminar architecture in AIPL1-LCA. (A) Cross-sectional OCT images along the vertical meridian through the fovea in a normal subject compared with six patients with AIPL1-LCA. Brackets defining the ONL and inner retina (left edge) and total retinal thickness (right edge) are labeled. ONL is highlighted in blue. (B) Thickness of the total retina (top), ONL (middle), and inner retina (bottom) along the vertical meridian in patients with AIPL1-LCA (n = 7). Patients are identified by symbols and grouped by age. Shaded areas: normal limits (mean ± 2SD) for retinal thickness (n = 27, ages, 5–58), ONL (n = 26, ages, 5–58) and inner retina (n = 14, ages 5–58). I, inferior retina; S, superior retina; F, fovea. Insets (top right corner): scan location.

Quantitation of thickness of the total retina, ONL, and inner retina in the AIPL1-LCA patients indicated a consistent pattern (Fig. 2B). At the fovea, there was no measurable ONL; eccentric to the fovea, the ONL in the patients was measurable but substantially reduced in thickness. At approximately 3- to 4-mm eccentricity into the superior and inferior retina, normal lamination was no longer present, and the patient scans had almost a bilaminar appearance with a thick vitread hyperreflective layer and a deep, thickened, hyporeflective layer. The thick superficial layer most likely includes the inner plexiform, retinal ganglion cell, and nerve fiber layers, whereas the deeper layer may be an amalgam of thickened INL with remnant photoreceptor nuclei. ^33,35 Total retinal thickness and inner retinal thickness were greater than normal in some of the younger patients. 

Topographic maps across an expanse of central retina illustrate the thickness of the ONL and inner retina in normal subjects and three patients with AIPL1-LCA (Fig. 3). In the normal retina (average map; n = 6; ages, 21–41), the ONL peaked centrally and declined with distance from the fovea; parafoveal thinning was more gradual in the superior retina (Fig. 3A, left). F2,P1 (age 16) retained a central island of abnormally reduced ONL with foveal loss (Fig. 3B, left). This island was surrounded by an undetectable photoreceptor layer. F6,P1 (age 33) also showed a central island of reduced ONL thickness with foveal loss and surrounding nonmeasurable ONL (Fig. 3C, left). F7,P2 (age 40) had a barely detectable ONL around a relatively larger central ONL loss than the other patients and an otherwise undetectable ONL (Fig. 3D, left). 

Figure 3.

 

Retinal thickness topography in AIPL1-LCA (A–D) Thickness topography of the ONL (left) and inner retina (right) mapped to a pseudocolor scale (shown beneath normal maps) in normal subjects (average map, n = 6; A) and three AIPL1-LCA patients (B–D). Insets (bottom right, inner retina) thickness difference maps showing the region that is within normal limits (white, defined as mean ± 2SD), or thickened (pink), compared with normal.

Inner retinal thickness topography in the normal retina had a foveal depression surrounded by an annulus of increased thickness, with a crescent of greater thickness extending toward the optic nerve (Fig 3A, right). The AIPL1-LCA patients showed increased inner retinal thickness compared with normal across most of the retina examined (Figs. 3B–D, right; insets). 

Rod-Mediated Function Retained in AIPL1-LCA

There was little or no measurable kinetic visual field in the AIPL1-LCA patients; only small islands of central or peripheral vision were detected with the V-4e target in three of the patients (Table 1). Full-field sensitivities to white stimuli in six patients (Fig. 4A, left) indicated that there was a range of sensitivity loss: three patients with light perception (LP) vision (F7,P2; F7,P1; and F4,P1) showed 7 to 8 log₁₀ units loss; two patients with hand motion (HM) vision (F2,P1 and F6,P2) had 4 to 5 log₁₀ units loss; and 1 patient with count fingers (CF) vision (F6,P1) had approximately 2 log₁₀ units of loss. In four of these patients, it was possible to perform chromatic full-field sensitivity testing, and all had evidence of retained rod function (Fig. 4A, right). 

Figure 4.

 

Visual function in AIPL1-LCA. (A) Left: loss of visual sensitivity by white FST in patients with AIPL1-LCA, in order of decreasing magnitude. Right: chromatic FST sensitivity differences (blue minus red) in patients with AIPL1-LCA. Results in normal subjects in the dark-adapted state (left) or at the cone plateau of dark adaptation after light exposure (right) served to define which photoreceptor mediates perception of each color (error bars, mean ± 2SD). Labels above the bars denote photoreceptor mediation: R, rods; M, mixed rod and cone; C, cones. Dashed lines: categorizations for R and C mediation. (B) Transient pupillary light reflex (TPLR) families of responses elicited by green (500 nm, black) stimuli over a range of intensities (−5.7 to 2.3 log₁₀ scot-cd · m⁻²) in a normal subject are compared with a representative AIPL1-LCA patient. Overlaid are traces (gray) evoked by scotopically matched orange (600 nm) stimuli. Stimulus steps were approximately 1 log unit. (★) Responses that reached the 0.3-mm criterion amplitude. Bottom left: stimulus monitor (0.1 second). (C) TPLR responses to the maximum stimulus intensity (green; 2.3 log₁₀ scot-cd · m⁻²) in AIPL1-LCA patients (n = 7) were compared to the normal response range (gray band = normal mean ± 2SD). (D) Summary parameters of TPLR response amplitude at a fixed (0.9 seconds) time (left) and TPLR threshold to a 0.3-mm criterion amplitude (middle). Gray symbol: normal mean ± 2SD. Chromatic TPLR response amplitude differences (green minus orange; right) measured from responses elicited with a scotopically matched pair of stimuli (B, arrows). Dashed lines: the range (mean ± 2SD) of TPLR response amplitude differences of the rod-dominated TPLR in normal subjects.

Pupillometry objectively confirmed the observation by full-field psychophysics of retained rod function. Families of TPLR responses elicited with brief stimuli in a representative normal subject are shown for comparison with those from a patient with AIPL1-LCA (Fig. 4B). In the normal subject, the TPLR threshold was near −4 log₁₀ scot-cd · m⁻². With increasing stimulus luminance there was an increase in amplitude and shortening of the latency to reach a 0.3-mm criterion response, and a retardation of the dilation phase. For each intensity, TPLR responses to the green stimulus (Fig. 4B, black traces) were nearly identical with those elicited by the scotopically matched orange stimulus (Fig. 4B, gray traces), supporting rod mediation of the normal dark-adapted TPLR. The response family in patient F6,P1 illustrates the abnormalities encountered in AIPL1-LCA (Fig. 4B). The TPLR threshold response in the patient was elevated by ∼3 log₁₀ units (to about −0.6 log₁₀ scot-cd · m⁻²) compared with that of the normal subject. With increasing stimulus intensity, amplitude increased, latency was shortened to criterion response, and the dilation phase was retarded. At the highest intensity (+2.3 log₁₀ scot-cd · m⁻²) TPLR response amplitude only reached ∼60% of normal (Fig. 4B). At each stimulus intensity, responses in the patient resembled normal responses evoked by a ∼3-log₁₀-units dimmer stimulus, indicating that loss of sensitivity may explain, at least in part, the differences in TPLR kinetics between the patient and the normal subject. As in the normal subject, pairs of responses elicited by scotopically matched green and orange stimuli were similar, supporting the notion that the responses are mediated by a rod photoreceptor mechanism and confirming the rod mediation determined by psychophysics in this patient (Fig. 4A). 

The TPLR was explored in six other patients, including some of the youngest subjects from our patient population (F1,P1 at age 1; F1,P2 at age 5). Response waveforms elicited with the maximum stimulus intensity summarize the results (Fig. 4C). Three of the patients did not show a TPLR response at maximum stimulation with either green or orange (not shown) stimuli; these were the patients (F4,P1; F7,P1; and F7,P2) with the greatest sensitivity loss (>7 log₁₀ units), shown by psychophysics. Four other patients showed responses that, although smaller in amplitude and slower in kinetics, could be similar to the normal waveform (Fig. 4C; gray band showing the normal mean ± 2SD). The maximum TPLR amplitude for the responding patients ranged from 27% to 81% of mean normal values (Fig. 4D). TPLR thresholds were greatly elevated with sensitivity losses ranging from 3.8 to 5.1 log₁₀ units (Fig. 4D). Next, we evaluated the photoreceptor mechanism mediating the TPLR in the patients. The differences in the TPLR response amplitude evoked by green or orange flashes were used as a quantitative metric to define the range in normal subjects, wherein responses are expected to be dominated by rods (Fig. 4D). All four responding patients fell within the range of differences observed in normal subjects, supporting a functionally intact TPLR pathway driven by an insensitive rod photoreceptor system. 

Phenotype of a Patient with Later-Onset Retinal Degeneration and AIPL1 Mutations

Patient F8, P1 was first evaluated at age 45 years and gave a history of reduced visual acuity, nyctalopia, and abnormal peripheral vision from the first decade of life. A clinical diagnosis of retinitis pigmentosa had been at age 14 years. There was no family history of similar visual problems. Progressive visual loss (visual acuity and peripheral vision) was noted by the patient over ensuing decades. 

At age 45, there were no lens opacities, and there were rare cells in the vitreous. On fundus examination, the macular area had relatively preserved RPE, and there was a bull's-eye–like lesion in the foveal region. There was pigmentary retinal degeneration with atrophy extending from the arcades into the midperipheral retina and attenuated retinal vessels; eccentric to this was better-preserved RPE. Optic nerve drusen were present in both eyes. The fundus appearance had not changed at age 49 (Fig. 5A). At age 67 years, NIR-REF imaging showed a large foveal region of depigmentation surrounded by a perifoveal annulus of pigmentation (Fig. 5B). Surrounding the macula was another annulus of depigmentation resulting in visibility of choroidal vasculature. A midperipheral ring of dense bone-spicule pigment surrounded the central region starting at eccentricities ranging from ∼6 mm (inferiorly) to ∼9 mm (nasally). NIR-RAFI results were consistent with NIR-REF: retinal regions of high choroidal reflectivity due to demelanization of the RPE showed low NIR-RAFI signal and the perifoveal ring of retained RPE pigmentation showed a relatively increased NIR-RAFI signal. Bone-spicule pigment was highly autofluorescent. SW-RAFI results demonstrated retained lipofuscin within the perifoveal region and hyperautofluorescence associated with optic nerve drusen (Fig. 5B). Cross-sectional imaging by OCT was similar to the results from the AIPL1-LCA patients; there was a deep foveal depression, loss of foveal ONL, and signs of retinal remodeling in the macular region (Fig. 5C). Topographic mapping of ONL showed some retained macular photoreceptors and a patch of photoreceptors superior to the optic nerve; the inner retinal map was increased in thickness at regions temporal and superior to the fovea but normal in thickness nasal and inferior to the fovea (Fig. 5D). 

Figure 5.

 

Phenotype of a patient with a later-onset retinal degeneration and AIPL1 mutations. (A) Color fundus photo of F8,P1 at age 49 years. (B) Digitally stitched NIR-REF, NIR-RAFI, and SW-RAFI images at age 67 showing the perifoveal ring of retained RPE melanin and lipofuscin visible on all three modalities, optic nerve drusen visible on SW-RAFI, and bone-spicule pigment visible on NIR-REF and NIR-RAFI modalities. Images are individually contrast-stretched for visibility of features. (C) Cross-sectional OCT image along the vertical meridian through the fovea. ONL, blue highlight. I, inferior; S, superior retina. (D) Thickness topography of the ONL and inner retina mapped to pseudocolor scales. Inset (bottom right, inner retina map): thickness difference map showing region that is within normal limits (white, defined as mean ± 2SD), or thickened (pink), compared with normal. (E) Kinetic perimetry results at ages 45, 60, and 67 showing progressive loss of visual field extent. (F) Static chromatic perimetry at age 49 demonstrating the remnant rod and cone function, centrally and in the far periphery. (G) Electroretinograms (ERGs) of a representative normal subject compared to the waveforms in the AIPL1-RD patient at age 49 showing remnant rod and cone function.

Visual acuities declined in both eyes in the interval from ages 45 to 67 (Table 1). Kinetic perimetry is shown in a sequence of fields of the right eye (Fig. 5E). At age 45, there was a central island of function (with both V-4e and I-4e test targets) separated from peripheral islands by an extensive absolute midperipheral scotoma. The central island was slightly reduced in extent by age 60, and I-4e detection was no longer present; peripheral islands diminished inferiorly. At age 67, there was no detectable central function and only minimal detection of the stimulus in the periphery. Chromatic perimetry at age 49 identified severely reduced cone and rod sensitivity in the central and peripheral islands (Fig. 5F). ERGs showed residual but much diminished rod and cone function (Fig. 5G; ∼90% loss of amplitude versus mean normal ^27,44 ). 

Discussion

The stepwise progress in the RPE65 form of LCA from gene discovery to increased understanding of molecular mechanism to proof-of-concept studies in animal models and then to human clinical trials (reviewed in Ref. 4) is a valuable template for therapeutic strategies of other incurable autosomal recessive human progressive retinopathies. One of the many keys to clinical trial success in RPE65-LCA has come from understanding the relationship between the animal models and the human disease expression. ^17,45 Even though the human RPE65-LCA disease at early stages shows considerable retinal degeneration compared with young animals, there is sufficient photoreceptor and RPE integrity to make treatment feasible. It is also important that there is detectable retinal structure and function in these patients well into the third decade of life. ^17,39,46,47  

The AIPL1 form of LCA has already progressed through many of the steps toward a treatment strategy in humans. Gene identification in relationship to the human disease ^18,19 has been followed by clinical characterizations ^{20 –23} and studies of mechanism. ^{48 –53} AIPL1 is shown to be expressed during both rod and cone development, and it plays a role in photoreceptor viability in the mature retina. ^54,55 An early hypothesis was that the Aipl1-deficient mouse is a phenocopy of the rd/rd (PDE6β-deficient) mouse, based on the rapidity of retinal degeneration common to the two models and defects found in rod cGMP phosphodiesterase (PDE) biosynthesis in Aipl1-deficiency. ^49,50 A role of AIPL1 in adult rods and rod phototransduction was originally found, ^{49 –51,54} but, more recently, a maintenance function of AIPL1 in adult cones has also been confirmed. ⁵⁵ It is now considered that AIPL1 is necessary for the assembly of PDE6β, specifically the catalytic subunit (α) of PDE6β. ⁵³ Further confirmation of the essential nature of AIPL1 in rods and cones is the proof-of-concept study that targeted and rescued both photoreceptors in Aipl1-mutant mice. ¹²  

Our human studies of AIPL1-LCA indicate an early retina-wide absence of rod and cone photoreceptor function by ERG, consistent with LCA in general and previous reports of the AIPL1 phenotype specifically. ^{20 –23} This finding could be due to loss of rod and cone photoreceptor cells or to dysfunction, such as would be postulated for a phototransduction defect. The absence of detectable ONL in most of the wide expanse of central retina that we studied supports cell loss as the basis for the severity of visual loss in the human disease. A question that cannot be answered in murine retinas lacking a fovea is the cause of the profound visual acuity disturbance in these patients. OCT cross sections through the central retina indicate loss of the cone-rich foveal ONL. The same level of visual acuity is present in many CEP290-LCA and LCA5 patients, but there is retained foveal ONL, emphasizing different mechanisms for low visual acuity in LCA patients with different genotypes. ^33,56 Of further interest, the foveal pit shape in AIPL1-LCA patients is reminiscent of a stage of foveal development before the centripetal movement of cones occurs to form the normal thick central layer of cone cell nuclei. ^{57 –60} An impact on human cone and rod development may thus be an important part of the disease expression in AIPL1-LCA; there is also likely to be further loss of remaining cells due to the photoreceptor maintenance function of AIPL1 in adulthood. 

Comparison of AIPL1-associated disease with autosomal recessive RP due to mutation in the PDE6B gene indicates dramatic differences, ⁶¹ thus confirming the impression from murine studies that Aipl1-deficiency is not a simple phenocopy of a rod-only phototransduction defect with secondary cone loss. ⁵⁵ In contrast to the profound and early visual acuity loss in AIPL1-LCA, PDE6B-RP shows persistent and excellent visual acuity. The residual visual sensitivity in the AIPL1-LCA patients is rod-mediated (suggesting remaining functioning rods in the retina) whereas chromatic perimetry and FST (SGJ, unpublished data, 2007) in PDE6B-RP are cone-mediated only. Both diseases, however, share the feature of inner retinal remodeling, suggestive of a severe loss of photoreceptors early in life. ⁶¹  

The patient with AIPL1 mutations and a later-onset protracted course of disease (F8,P1) deserves discussion. Screening of a large cohort of retinal degeneration patients after the AIPL1 gene was discovered led to the observation that two probands from different families had a heterozygous 12-bp deletion in AIPL1, not found in control subjects. The clinical diagnoses were cone–rod dystrophy (CRD, one family with a single affected member) and juvenile RP (two affected members separated by a generation). ¹⁹ Phenotype details were not provided. To our knowledge there are no further reports of diagnoses other than LCA associated with AIPL1 mutations until the present study. Also of interest is that F8,P1 was the only individual in the current group of patients with a mutation in the first of three tetratricopeptide (TPR) motifs in the AIPL1 molecule. One other disease-associated change has been reported in this domain (Q141H ⁶² ) in an LCA patient, but no phenotype information is available. One explanation for the phenotype in F8,P1 is that mutations in this TPR domain cause a milder retinal degeneration. 

The presentation of F8,P1 was one of a widespread retinal degeneration with some degree of maculopathy at age 45. The recordable ERG showed relatively equal loss of rod and cone signals and would conform to our previous ERG definition of a CRD. ⁶³ There was some preserved central cone sensitivity and some peripheral rod function. Whether the clinical diagnosis is AIPL1-RP or AIPL1-CRD at this stage of our understanding becomes a nomenclature debate, until more patients are identified and studied. With two disease alleles (one of which is the common W278X variant) and no history of other generations affected, we assume this is autosomal recessive disease. It would be of strong interest if the proband from the previous report of dominant disease in a single family ¹⁹ could be screened for a possible second disease-causing allele. 

Why is this single case important? The severity of human disease in AIPL1-LCA suggests that unless there is evidence provided by future studies of much younger patients that would show measurable photoreceptors beyond the small parafoveal ring of very reduced ONL, then AIPL1-LCA may not be a candidate for gene augmentation therapy despite the encouraging proof-of-concept success in mice. ^10,11 The relatively slower disease of AIPL1-RD, however, and its model of an Aipl1 hypomorph ⁴⁹ may be a far better target for treatment. Identification of these patients through screening large cohorts of molecularly unclarified patients with the clinical diagnosis of RP or CRD seems a worthy goal, considering the progress to date toward therapy. 

Footnotes

 Supported in part by the Foundation Fighting Blindness, Hope for Vision, Macula Vision Research Foundation, the Grousbeck Family Foundation, The Chatlos Foundation, and The Yedidut Research Grant.

Footnotes

 Disclosure: S.G. Jacobson, None; A.V. Cideciyan, None; T.S. Aleman, None; A. Sumaroka, None; A.J. Roman, None; M. Swider, None; S.B. Schwartz, None; E. Banin, None; E.M. Stone, None

The authors thank Anat Blumenfeld and Dror Sharon for molecular studies and Melani Oliveras and Elaine Smilko for critical administrative help.