Long-Term Structural and Functional Assessment of Doyne Honeycomb Retinal Dystrophy following Nanosecond 2RT Laser Treatment: A Case Series

Doyne honeycomb retinal dystrophy (DHRD) or autosomal dominant radial drusen (Malattia Leventinese, OMIM#126600, ORPHA:75376) is an autosomal dominant disorder caused by a single missense variant (Arg345Trp or R345W) in the epidermal growth factor (EGF)-containing fibulin-like extracellular matrix protein 1 (EFEMP1) gene (2p16.1) [1‒4]. Drusenoid deposits involving the posterior pole and peripapillary area, often with a radial distribution, are typical manifestations of the disease. Molecular and clinical evidence indicates that pathogenic variants of EFEMP1 gene may alter the extracellular matrix in Bruch’s membrane, leading to the accumulation of basal laminar deposits [1, 3]. Indeed, optical coherence tomography (OCT) shows diffuse alterations of the retinal pigment epithelium (RPE)/Bruch’s membrane complex with relative sparing of the neurosensory layers [5‒7]. Also mutations in the gene encoding tissue-inhibitor metalloproteinase-3 (TIMP3) may be associated with a severe form of DHRD [8‒10], although TIMP3 mutations are most commonly associated with Sorsby’s fundus dystrophy [11]. In intermediate age-related macular degeneration (AMD), drusen or drusenoid deposits, Bruch’s membrane thickening, and RPE atrophy (see reviews [12, 13]) are observed, similar to DHRD. Mouse models of both AMD and DHRD show complement activation and RPE atrophy (quoted in [12, 13]). Lenassi et al. [14] showed that low-energy argon laser treatment induced drusen clearance and improved visual function in DHRD patients with a confirmed EFEMP1 mutation [14]. A low-energy subthreshold nanosecond laser, 2RT™ (Ellex, Adelaide, SA, Australia), has been employed to cause controlled RPE injury without significant damage to the neurosensory retina or gliosis [15‒17]. A clinical trial on intermediate AMD provided evidence of efficacy at the 12-month follow-up, with improvement in retinal function and reduction in the mean drusen area [18]. A similar effect was found in the ApoE-null mouse model of AMD [19], with thinning of Bruch’s membrane and increased expression of matrix metalloproteinases-2 and -3 following 2RT treatment. A randomized trial in early/intermediate AMD patients confirmed the potential efficacy of 2RT [20].

Based on preliminary evidence of 2RT safety and efficacy in DHRD, and the similarity in the pathophysiology of DHRD and AMD, we treated 3 patients with DHRD caused by the EFEMP1 pathogenic variant, using a published 2RT protocol [21]. The treatment protocol and short-term effects of 2RT in one of these patients were described in a previous study on 1 patient by our group [21]. The aim of the current study was to report, for the first time, the long-term clinical findings in the same patient and in two additional DHRD patients following 2RT treatment.

Three DHRD patients (one male and two sister females, aged 41–46) with pathogenic variants in EFEMP1 gene and drusenoid deposits at the posterior pole were examined at baseline and at 2–4 months intervals up to 30 months following 2RT treatment. All 3 patients underwent one or two treatment sessions in one or both eyes during the first 12 months of follow-up. One patient was treated only in the left eye (LE) (case 3). Each patient underwent a full ophthalmologic examination, spectral domain OCT, central perimetry with frequency doubling technology (FDT; equivalent in area to Humphrey visual field 30-2), and mesopic and photopic full-field electroretinograms (ERGs) as well as multifocal ERGs, recorded at baseline and 12 months after treatment. FDT perimetry is based on the flicker perception generated by counterphase flickering of a low spatial frequency grating modulated at a high temporal frequency [22]. Although the FDT 10-2 protocol might have been more sensitive, the 30-2 field was employed to better evaluate extramacular sensitivity.

Genomic DNA was extracted from buccal swab [23] using the MagPurix Forensic DNA Extraction Kit and MagPurix Automatic Extraction System (Resnova), according to the manufacturer’s instructions. The concentration and quality of the extracted DNA were determined using a DeNovix Spectrophotometer (Resnova). The extracted DNA was sequenced using NextSeq 550 (Illumina), and library preparation was performed on 20–50 ng/μL of DNA using Illumina (Resnova) DNA Prep with Enrichment and Tagmentation according to the manufacturer’s instructions. The obtained libraries were sequenced at 2 × 100 bp, and the sequencing quality of the resulting data was expected to reach a quality score of >30 (Q30) for ∼80% of the total called bases. For the resulting variants, only those reporting a minimum coverage of 20X were considered eligible for bioinformatics analysis. Moreover, the functional annotation of detected variants was performed using BaseSpace Variant Interpreter v. 2.15.0.110 (Illumina) and the wANNOVAR tool (https://wannovar.wglab.org/). Genetic variants were interpreted using publicly available reference databases (ClinVar, 1000 Genomes, GnomAD, and VarSome). Variants were classified and clinically interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines [24], using the VarSome online platform (https://varsome.com). According to the ACMG guidelines, c.1033C>T can be classified as a pathogenic variant, considering that it is absent in gnomAD genomes and gnomAD exome databases (PM2), and multiple lines of computational evidence support a deleterious effect on the gene or gene product (PP3); reputable source (ClinVar, UniProt Variants, VarSome) recently reported variants as pathogenic (PP5). Moreover, ClinVar and UniProt Variants are associated with DHRD. The identified variant was confirmed by direct sequencing performed with a BigDye Terminator v3.1, BigDyeX Terminator, and ABI3130xl (Thermo Fisher) according to the manufacturer’s instructions.

The genetic evaluation study was approved by the Ethics Committee of the Santa Lucia Foundation (CE/PROG.650 approved on March 1, 2018). This study adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all 3 patients for both 2RT treatment and publication of their anonymized clinical findings and related images after the aims of the study were explained in detail.

A male Caucasian patient (from a pedigree different from that of cases 2 and 3, see below) aged 35 years had a moderate loss of visual acuity in the LE (20/40) and normal right eye (RE) acuity (20/20). The baseline clinical findings of this patient were the object of a previous publication [21]. Fundus examination revealed typical DHRD lesions. The central macular thickness on OCT was normal in both the eyes. Central perimetric sensitivity by FDT showed an abnormal standard deviation pattern in both eyes, indicating localized defects. Full-field mixed (3.0 cd/m²) and photopic ERGs (single flash, light adapted at 3.0 cd/m²) as well as multifocal ERGs were reduced compared to the normal values in both eyes. Genetic analysis revealed a common heterozygous DHRD variant (c.1033C>T; R345W) in EFEMP1 (2p16.1).

Female Caucasian patient, 36 years of age, sister of case 3, with normal visual acuity but complained of blurring of central vision and reduced contrast perception. Fundus examination revealed typical DHRD lesions. The central retinal thickness was normal in both eyes. Central perimetric sensitivity by FDT showed generalized depression in both the eyes. Full-field ERG amplitudes were normal in OD and reduced compared to normal in OS. Multifocal ERGs were abnormally reduced in both eyes. Genetic analysis revealed a heterozygous DHRD variant (c.1033C>T; R345W) in EFEMP1 (2p16.1).

Female Caucasian of 46 years of age, sister of case 2, had moderate visual acuity loss (OD 20/40, OS 20/25) and typical DHRD fundus lesions. Central retinal thickness was normal in both eyes, whereas central perimetric sensitivity at FDT was severely reduced in both eyes (OD MD, −12 dB; OS MD, −9 dB). The full-field ERGs had normal amplitudes in both eyes. Multifocal ERGs were abnormally reduced in both eyes. Genetic analysis revealed a heterozygous DHRD variant (c.1033C>T; R345W) in EFEMP1 (2p16.1).

All patients underwent one or two 2RT sessions. Case 1 had two treatments, the sisters case 2 and 3 had only one session. In case 1, the two sessions during follow-up were temporally separated by at least 12 months. The treatment followed a standardized protocol [21]. Briefly, ultralow-energy laser pulses were applied in 12 spots around the macula of one eye (0.15–0.45 mJ), using 400 μm diameter spots, 3-ns pulse length, 532 nm wavelength, and energy titrated to the patient. Patients 1 and 2 were treated in both eyes, whereas patient 3 was treated with LE only, following her preference (and despite the LE vision was better than that of RE, see below Table 1). None of the patients experienced treatment-related adverse events or unanticipated events.

The long-term results obtained for the 3 patients are presented in Table 1. None of the patients had treatment-related side effects. Compared to baseline findings, visual acuity improved by 2–10 letters in both eyes of case 1 and LE of case 2, while it moderately decreased (by 6 letters) in the RE of case 2 and LE of case 3; perimetric sensitivity was stable in case 1 and improved substantially in both eyes of cases 2 and 3; mixed rod-cone ERG amplitude increased significantly in 2 patients (cases 1 and 2) and declined slightly in case 3, and the cone b-wave had a similar behavior.

Representative mixed rod-cone ERGs and cone ERGs recorded at baseline and 1 month after 2RT in case 3 are shown in Figure 1. It can be noted that while the mixed rod-cone b-wave decreased slightly from the baseline, the cone b-wave amplitude increased. Further ERG recordings of this patient during follow-up did not show substantial changes.

The perimetric FDT maps recorded in the same patient (case 3) at baseline and 1 year after 2 RT are shown in Figure 2. It can be noted that FDT sensitivity improved substantially from baseline in both eyes at 1 year, indicating improved contrast sensitivity.

In Figure 3, plots of multifocal ERG response amplitude densities as a function of retinal eccentricity, recorded at baseline and after 12 month following treatment, are shown. No significant changes in response amplitude densities were observed in any of the patients.

The OCT central macular thickness was stable in all cases. The retinal microstructure highlighted by OCT was compared before and 12 months after 2RT for both eyes in each case (Fig. 4a, c). In all study eyes, at 12 months, drusenoid deposits tended to be more thickened at the level of the RPE, though their character changed. In addition, RPE atrophic changes appeared to progress. The retinal thickness did not change significantly in any of the study eyes.

All 3 patients reported subjective improvement in their visual function, with special reference to contrast perception. Although contrast sensitivity was not systematically recorded in these patients, FDT sensitivity in the central 30° provided an indirect but related measure of contrast perception to high temporal frequency, low spatial frequency patterns [22]. The CARE checklist has been completed by the authors for this case report, attached as online supplementary material (for all online suppl. material, see https://doi.org/10.1159/000534579).

The present study reports the long-term retinal functional and structural changes in 3 patients with DHRD who underwent one or two sessions of nanolaser 2RT treatment over 12 months. To our knowledge, this is the first report of the long-term results of 2RT treatment in DHRD. Currently, no treatment is available for these orphan diseases. It should also be noted that the natural history of DHRD predicts long-term decline in visual acuity, photoreceptor degeneration, and subretinal and intraretinal fluid accumulation due to RPE dysfunction [7]. Interestingly, the phenotype may be similar to that of TIMP3-related Sorsby’s dystrophy [11]. In our patients, one or two sessions of 2RT laser treatment performed over 12 months stabilized or improved some parameters of retinal function, such as perimetric sensitivity and full-field ERG amplitudes. At 24 months, a moderate decline in visual acuity was observed in 2 patients (Table 1), in one of them mainly due to intraretinal fluid accumulation leading to cystoid macular edema that required treatment with carbonic anhydrase inhibitors. The late changes, together with choroidal neovascularization leading to subretinal or intraretinal fluid accumulation, are likely related to the natural history of the disease and may have been prevented by further 2RT treatments.

As previously hypothesized [21], the long-term improvement in retinal function after treatment supports improved photoreceptor function due to an improved rate of retinoid recycling and/or RPE rescue. Some sensitivity improvement was observed in the fellow untreated eye (see Fig. 2). It is interesting to note in this respect that similar effects were observed in our previous study [21] and by Jobling et al. [19], suggesting an indirect induction effect exerted by this subthreshold laser at systemic level. These effects may primarily involve the extramacular retina since treatment did not involve directly the macula butonly the spots around the macula (see Methods). This may explain the increase in amplitude from the baseline of full-field ERGs, found in 2 patients. Full-field ERGs improvement may be indicative of improved retinoid recycling, leading to increased visual sensitivity, especially in rods. By contrast, multifocal ERG as a measure of macular cone photoreceptors did not show significant changes at 12 months following treatment, supporting a rod-specific effect. We cannot exclude that the full-field ERG changes observed in 2 patients may merely reflect a temporal fluctuation, without significant decrease from baseline, confirming the safety of the procedure at level of the outer retina.

A limitation of this case series report is the very small sample size. Further studies in patients with DHRD evaluating recovery after bleaching of retinal sensitivity after 2RT laser therapy are required to support our hypothesis that rods may improve functionally as a result of improved retinoid recycling. Nonetheless, the observed 2RT effects are consistent with the mechanism of action proposed for this treatment, which in cultured RPE was able to target the RPE both in vitro and in vivo, causing debridement of the cells and consequent stimulation of a wound-healing response leading to layer reformation [25]. Since DHRD is a genetic-based dysfunction of retinoid recycling and some benefits were seen from only one or two sessions of 2RT laser, repeated laser sessions could temporarily stimulate local clearance adding safely further benefits. Clearly, the enzymatic dysfunction underlying retinal dystrophy would continue to progress with altered phototransduction.

In conclusion, the present case series report adds new information on the long-term effects of 2RT laser treatment on retinal structure and function in DHRD, particularly supporting the safety and potential efficacy of this treatment for this rare and orphan disease of the retina. Further randomized clinical studies are required to fully assess the efficacy of treatment for visual function in DHRD.

The molecular genetic study protocol was reviewed and approved by the Ethics Committee of the Santa Lucia Foundation (CE/PROG.650 approved on March 1, 2018). Written informed consent was obtained from the patients for publication of the details of their medical case and any accompanying images.

The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.

The study was supported by a grant of the Macula and Genoma Foundation.

A.C. and B.F. contributed to the design and implementation of the research, A.C., B.F., M.D., J.S., F.D., J.H.L.G., R.C., and E.G. to the analysis of the results and to the writing of the manuscript. A.C. conceived the original and supervised the project.

Andrea Cusumano and Benedetto Falsini contributed equally to this paper.

All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author.