Introduction

A subset of genetically related autosomal dominant drusen (OMIM 126600) were originally described as Doyne honeycomb retinal dystrophy in Britain and as malattia leventinese (MLVT) in Switzerland, which are caused by a single missense mutation Arg345Trp in the EFEMP1 gene.1 Characteristic signs are early onset drusenoid deposits at the posterior pole and in the peripapillary area with a possible radial distribution, increasing confluence with age and inter- and intrafamilar variability.2 Fu et al.3 suggested that mutant Efmp1 protein might change the extracellular matrix in Bruch's membrane resulting in basal laminar deposits. Retinal layer characterization, using time-domain optical coherence tomography (OCT), has demonstrated diffuse alterations of retinal pigment epithelium (RPE)/Bruch's membrane complex4, 5 with preservation of the neurosensory layers.5 Detailed analyses, in particular of the outer retinal layers have, however, been limited by the image resolution of commercial time-domain instrumentation. Here, we demonstrated significant photoreceptor changes in two cases with variable EFEMP1 associated retinal dystrophy.

Methods

Two patients diagnosed with the MLVT phenotype and identified c. R345W mutation in the EFEMP1 gene (Carver laboratory, Iowa, IA, USA) were recruited through the Ocular Genetics Clinic at the Hospital for Sick Children in Toronto, Canada. Written informed consent was obtained from patients. The project was approved by the Research Ethics Board at Sick Kids as well as the University of California, Davis Institutional Review Board, and conducted in accordance with the Tenets of Helsinki. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research.

Vision function assessment included best-corrected monocular near and distance visual acuity, colour vision and contrast sensitivity. Retinal responses were recorded using full-field electroretinography (ffERG; International Society for Clinical Electrophysiology of Vision (ISCEV) standard)6 and multifocal electroretinography (mfERG; 103 hexagons, ISCEV recommendation).

Retinal image acquisition was achieved using a custom built high-speed, high-resolution Fd-OCT system7 (axial resolution: 4.5 μm; acquisition speed: 9 frames −1s, 1000 A scans s−1) constructed at UC Davis with a sample arm scanning head (Bioptigen Inc.). Horizontal scans of 6 mm length or a volumetric scan series permitting image acquisition over an area of 6 × 6 mm and OCT fundus reconstruction were registered through the macular area. Raw image data were post-processed and retinal layers were identified as described earlier (Figure 1).8, 9

Figure 1
figure 1

Six-millimetre horizontal Fd-OCT scan through the right macula of a 36-year-old control. CL, connecting cilia; GCL, ganglion cell layer; ILM/NFL, internal limiting membrane/nerve fiber layer; INL, inner nuclear layer; IPL, inner plexiform layer; ISL, inner segment layer; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; OSL, outer segment layer; RPE/BM, retinal pigment epithelium/Bruch's membrane; VM, Verhoeff's membrane.

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Results

A 33-year-old woman (case 1) and her 60-year-old asymptomatic mother (case 2) of French/Scottish/Dutch ancestry were assessed. Results of vision function are summarised in Table 1. Fundus examination showed small, round, yellowish-white deposits throughout the macular and peripapillar area with perifoveal pigmentary changes and RPE atrophy (case 1) (Figure 2). The mother's fundus revealed two areas of extrafoveal confluent white deposits with central RPE atrophy and few retinal deposits in the nasal fundus in both eyes. (Figure 3).

Table 1 Vision function results of the two patients with EFEMP1 mutation
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Figure 2
figure 2

Composite of fundus photograph and serial horizontal Fd-OCT scans (6 mm) of the right and left eyes of patient no.1. The locations of OCT B-scans are denoted by lines (a–e) shown on the OCT fundus image (intensity projection of the OCT volume), which is superposed and registered to the colour fundus photo. Lower panel shows a virtual C-scan at the level of the photoreceptor inner/outer segment junction segmented from the reconstructed OCT volume. B-scans illustrate extensive deposits (denoted by a star in one scan) in the sub-RPE area with separation between the RPE and Bruch's membrane (arrow denotes the location in two scans). Circles provide example of disruption of the outer segment layer, inner segment layer, and outer nuclear layer.

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Figure 3
figure 3

Composite of colour fundus photograph and serial horizontal Fd-OCT scans (6 mm) and virtual C-scan (for description see Figure 2) of the right and left eye of patient no.2. The locations of OCT B-scans are denoted by lines (a–e) shown on the OCT fundus image. Circels denote areas of sub-RPE deposits with corresponding photoreceptor disruption. B-scans illustrate disruption of the outer retinal layers (star denotes area in three scans). Circles provide example of cone-shaped deposits in the RPE area extending into inner retinal layers. Subfoveal photoreceptor layers were preserved.

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Macular scans from case 1 demonstrated a separation of Bruch's membrane and RPE by extensive dense highly reflective material in the sub-RPE zone (Figure 2). It appeared that those deposits are pushing and elevating the outer retinal layers. Outer segment layer, inner segment layer, and outer nuclear layer were disrupted more in the subfoveal than in the extrafoveal areas. Outer limiting membrane structure was disturbed. Retinal microstructure showed similar but more focal changes in correspondence to the visible fundus abnormalities with preserved subfoveal layers in case 2 (Figure 3). Photoreceptor disruption was confined to areas with sub-RPE deposits in both cases.

Discussion

High-resolution retinal imaging permitted identification of disrupted photoreceptor layers in two patients with variable MLVT phenotype caused by the c. R345W mutation in the EFEMP1 gene for the first time. Our findings are in agreement with histopathological studies of MLVT cases, which demonstrated formation of hyaline substances between retina and choroid with extension through the outer limiting membrane in some cases. There, destroyed rod and cone photoreceptor layers and outer nuclear layer but unchanged inner retina layers were described.10, 11

Efemp1 knock-in mice showed deposits between the plasma and basement membrane of the RPE, which altered RPE cell ultra structure. Neurosensory retinal changes included outer segment shortening and detachment from the RPE. Older mice exhibited focal thickening of Bruch's membrane, choriocapillaris degeneration, and outer nuclear layer thinning.3, 12 Abnormal photoreceptor structure was evident in areas with diffuse or focal sub-RPE deposits in our patients. Increasing accumulation in the sub-RPE space might lead to altered RPE function and morphology, which might activate the complement system.3 Impaired RPE function will compromise photoreceptor survival and therefore visual function. Visualising of distinct retinal features using high-resolution imaging provides important information for better understanding of the disease process.