Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging.
One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL).
Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects.
This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.
Lafora progressive myoclonus epilepsy is a neurodegenerative metabolic disease caused by accumulation of insoluble polyglucosan aggregates. , These aggregates, termed Lafora bodies, build up throughout the body and central nervous system, including the neurosensory retina, and lead to progressive symptoms early in adolescence. Post-mortem investigations have found retinal cell loss and Lafora bodies within and around inner retinal neurons, predominantly in the ganglion cell and inner nuclear layers. , Standard fundus examination and visual acuity testing is often unremarkable, but electroretinographic findings reveal retinal dysfunction. The neural retina is accessible to noninvasive imaging and provides a window into the central nervous system. Noninvasive retinal imaging has been used to demonstrate retinal alterations in a number of neurodegenerative diseases. Here we used spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO) in three genetically confirmed individuals with Lafora disease to look for similar retinal structural changes.
Materials and methods
This study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board at the Medical College of Wisconsin. Informed consent was obtained from all subjects, or from their legally authorized representative. Axial length was measured in each eye using an IOL Master (Carl Zeiss Meditec, Dublin, CA, USA) for scaling retinal images. One drop of phenylephrine hydrochloride (2.5%) and tropicamide (1%) was administered to one eye to dilate the pupil and suspend accommodation for imaging.
High-resolution SD-OCT volumetric (nominal 6 × 6 mm scans, 512 A-scans/B-scan, 128 B-scans) and horizontal line (nominal 6 mm) scans of the macula were acquired using a Cirrus HD-OCT device (Carl Zeiss Meditec) and/or the Bioptigen Envisu R2200 SD-OCT system (nominal 7 mm scan; Leica Microsystems, Wetzlar, Germany). Mean retinal thickness within a 6 mm radius of the foveola was calculated using Cirrus’ built-in macular analysis software. Bioptigen line scans were registered and averaged as previously described. The OCT line scans were then semi-automatically segmented using the Duke Optical Coherence Tomography Retinal Analysis Program (DOCTRAP) software to obtain retinal thickness measurements of the ganglion cell-inner plexiform layer (GCIPL) [boundaries: bottom of the retinal nerve fiber layer (RNFL) to top of the inner nuclear layer (INL)] and the outer nuclear layer + Henle fiber layer (ONL+) [boundaries: bottom of outer plexiform layer (OPL) to top of the external limiting membrane] using custom MATLAB software (MathWorks, Natick, MA).
The foveal avascular zone (FAZ) and the vasculature surrounding the optic nerve head (ONH) were assessed in Case 3 using the AngioVue OCT-Angiography system (Optovue Inc. Fremont, CA). For each eye, multiple foveal (nominal 3 × 3 mm, 304 B-scans at 304 A-scans/B-scan) and ONH (nominal 4.5 × 4.5 mm; 400 B-scans at 400 A-scans/B-scan) scans were acquired and averaged together as previously described. For FAZ measures, data were extracted from the whole retinal vasculature slab (boundaries: internal limiting membrane (ILM) to 9 μm below the OPL). For ONH scans, data were extracted from the radial peripapillary capillaries slab (boundaries: ILM to bottom of RNFL). Area and acircularity of the FAZ, segmented by a single observer (R.E.L.), as well as ONH capillary density was measured and assessed as previously described.
Using previously described AOSLO systems, , confocal and non-confocal split-detection videos focusing on the photoreceptor layer or the RNFL were acquired at the macula and along the superior and nasal meridians. Individual videos were registered and averaged to produce single high-resolution images. The images were montaged together semi-automatically and regions of interest (150 × 150 μm) at 1° and 2° from the fovea were extracted. Cones were semi-automatically counted by a single observer (H.H.) and cone density was calculated using custom software (Translational Imaging Innovations, Inc., Hickory NC). GraphPad Prism (La Jolla, CA, USA) was used for statistical analysis.
A 24-year-old female was diagnosed with molecularly confirmed Lafora disease after presenting with typical symptoms (compound heterozygous EPM2A mutations: p.Asn163Asp & p.Ala254Metfs*33). Clinical ophthalmological findings have been previously published for this patient. There was overall retinal thinning at 3 and 6 mm from the fovea in both eyes ( Fig. 1 A). GCIPL and ONL+ thickness measurements across the macula were within the lower limits of the previously published normative range ( Fig. 1 B). Cone density estimates at 1° (~43,600 cones/mm 2 ) and 2° (~36,800 cones/mm 2 ) from the fovea were similar to control averages (average ± 2SD; 51,400 ± 13,300 cones/mm 2 and 32,500 ± 7500 cones/mm, respectively) with unremarkable and consistent cone topography ( Fig. 1 C). Within the RNFL near the ONH, and at the level of the ILM near the fovea, nummular reflectivity (i.e., small hyperreflective punctate structures ) was observed ( Fig. 1 D).
A 20-year-old female was diagnosed with molecularly confirmed Lafora disease after presenting with symptoms (compound heterozygous EPM2A mutations: p.Arg241Ter & p.Asn163Asp). Overall retinal thinning was observed across the macula ( Fig. 2 A). In both eyes, six of the nine regions of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid were in the lower 5th percentile for the normal distribution of retinal thickness, while one region (6 mm temporal) was in the lower 1st percentile. We were unable to acquire OCT line scans to assess GCIPL and ONL+ thickness in this patient. Parafoveal cone density estimates ( Fig. 2 B; 1°: ~32,800 cones/mm, 2°: ~27,000 cones/mm 2 ) were similar to control averages as previously published. We again observed nummular reflectivity within the RNFL approximately 8° nasal to the fovea ( Fig. 2 C).