Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss and retinitis pigmentosa (RP) in some subtypes. In addition, patients can present with vestibular and olfactory deficits (1). The syndrome is the most common genetic cause of deaf-blindness (1, 2) and was first recognized and described by Albrecht von Graefe in 1858 (3) and further investigated by Charles Usher in 1914. Functionally, RP is characterized by impaired night vision, progressive loss of the visual field and a reduced visual acuity due to the progressive degeneration of the photoreceptors. Fundus features include blood vessel attenuation, disc pallor, cystoid macular edema and peripheral bone spicule derived from the retinal pigment epithelium (RPE) changes (4). Usher syndrome is subdivided into four clinical subtypes (USH1, USH2, USH3 and USH4) depending on the severity and time of onset of auditory, vestibular and visual symptoms (5). At least 9 causative genes have been found and identified to be responsible for Usher syndrome: Five for USH1 (MYO7A, USH1C, CDH23, PCDH15, and USH1G), three for USH2 (USH2A, ADGRV1, and WHRN) and one for USH3 (CLRN1). However, other genes and loci have been proposed and discussed to be related to Usher syndrome (6).

Hearing aids or cochlear implants can compensate the hearing loss, but so far there is no approved causal genetic treatment for the RP in Usher syndrome. However, over the last several years research on Usher syndrome has increased, with studies on gene and cell therapies providing hope for an efficient prevention of the sensory loss (7). Nevertheless, more studies on the natural history of the disease are required to aid planning clinical trials. Reliable and sensitive tools need to be tested and developed to evaluate the effect of experimental treatments.

With the availability of adaptive optics (AO) fundus imaging, the non-invasive visualization of the photoreceptor mosaic on a cellular level became possible (8, 9), and abnormalities in individual cone photoreceptor mosaic in patients with various retinal diseases could be studied (10–18). AO technologies have also been combined with optical coherence tomography (AO-OCT), fundus photography and scanning-laser ophthalmoscopy (AO-SLO), that increased the sensitivity and resolution and thus provided new insights into the effects of retinal degenerations (12–14, 18–24).

In this study we used flood illuminated adaptive optics (FI-AO) camera (rtx1™), an advanced high-resolution fundus imaging method to quantify the photoreceptor mosaic in RP associated with Usher syndrome. Additionally, we compared and correlated the results with those of spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence photography (FAF), semiautomatic kinetic visual field testing, full-field (ffERG) and multifocal electroretinography (mfERG) that are used for clinical ophthalmological diagnostics in our clinical routine for inherited retinal dystrophies. The aim was to explore the use of high-resolution FI-AO for diagnosis and monitoring of disease progression.

Ten patients with a clinical diagnosis of RP with Usher syndrome and likely biallelic variants in MYO7A (Usher syndrome type 1) or USH2A (Usher syndrome type 2) who presented for follow-ups at the University Eye Hospital, Tübingen were included in the study ( Table 1 ). Exclusion criteria included the inability of a moderate fixation, macular edema, central media opacity such as but not only cataract. In addition, patients with a history of any systemic disease (e.g. hypertension) or other eye complications were excluded. The examinations were approved by the institutional Ethics Committee and adhered to the Declaration of Helsinki. All subjects were informed about the consequences of the procedure and informed consent was received from all subjects. Statistical analysis was performed using SPSS (Version 27.0 SPSS Inc. Chicago, IL) and the graphs were created using JMP16 (SAS institute Inc., Cary, NC, USA).

The clinical characteristics and genetic profiles of the patients are summarized in Table 1 , along with a short description of the fundus photography, imaging and ERG results. The patients were between 16 and 60 years old ( Table 1 ). All USH1 patients had cochlea implants and USH2 patients had hearing aids. The best-corrected visual acuity (BCVA) ranged from 20/400 (feet) to 20/20 (feet). One eye had light perception. Here, only limited examinations were possible. The mean BCVA was 0.25 ± 0.43(logMAR) for the right eye and 0.24 ± 0.52 (logMAR) for the left, which is approximately equivalent to 20/40.

Our results show that the cone density in patients with RP related to Usher Syndrome using the adaptive optics flood illumination retina camera was decreased compared to previously published data on healthy eyes (27). Photoreceptor cell death was seen as a low cone density, along with disorder of the cone mosaic ( Figures 4 , 5 ). It is known that the cones within the central 2 degrees area cannot be resolved with AO due to the limit of resolution of the device. However, because a central loss of cones is expected in RP patients with Usher Syndrome, making the remaining cones detectable, the visualization and measurement of cell density could be established in the central fovea and at 1 degree. Therefore, we could observe visible cones in the fovea in the early stages of the disease, as well as a loss of cones outside the fovea, seen as blurry, dark patches. However, due to the anatomical distribution of the cones (28), we were expecting a much higher cone density in the central area, especially in the patients with a still good preserved visual acuity.

There are other reports that have examined the cone mosaic in RP using AO with similar patterns found as here (13, 29, 30). Gale et al. (29) categorized the changes in cone reflectivity into hypo-reflective blurred cone-like structures, higher frequency disorganized hyper-reflective spots and lower frequency hypo-reflective spots, which mirror progressive phases of cone degeneration observed using SD-OCT and FAF, as also shown by Tojo et al. (30) and Ueno et al. (31). This was also obvious in our study: The blurry patches and cone density matched the cone photoreceptor degeneration patterns shown by the SD-OCT and FAF imaging ( Figures 2 , 3 ). Gale et al. (32) have also shown that the repeatability of the results in RP patients is the same as that of control subjects.

High-resolution measurements of the cone mosaic performed with the confocal AOSLO (17, 33–36), which measures aberrations from the entire scanned area of the retina including the fovea, confirm these findings. Even if the outer retinal architecture in the SD-OCT is normal, the cone density has been shown to range from normal to severely reduced in RP patients (17, 19, 33, 34). A study investigating RP patients with Usher syndrome has further shown that the foveal cone density determined with an AO-SLO can be decreased before visible changes in the SD-OCT or a decline in visual function (36). This highlights the sensitivity of the high-resolution AO imaging and lends weight to studies demonstrating that visual acuity and sensitivity measurements are relatively poor markers for the severity of retinal degeneration compared to high resolution imaging of foveal cone mosaic (11).

Nevertheless, one of our limitations is that when using the rtx1 compared to an AOSLO, the cone density may be underestimated due to poor image quality or reflectivity of the cones. It is not possible to distinguish between not waveguiding cells and dark spots. Furthermore, due to the lack of information on patients’ eye length, the cell density could only be expressed using cones per degree² (visual units).

Moreover, in this study, retinal areas within the hyperautofluorescence in FAF image indicated normal outer retinal structures, whereas the AO-FIO images displayed increased spacing in the cone mosaic, as previously found with, AO-SLO imaging (26) and in descriptive analysis (15).

The ERGs were extinguished in 7 patients and reduced in 3 patients confirming the report by Stingl et al. (37) that the ERG is abnormal in Usher patients. Our results revealed a correlation between the mfERG results and the other imaging techniques. The mfERG has been shown to reliably provide even small remaining responses in the macular region of RP patients (38–40) and was also reduced in all our Usher patients. This study could confirm that the cone density of the central retina was in most cases significantly correlated ( Table 1 ) to the mfERG response in the central 3° of the retina. When the cone density measurements from AO retinal images were compared with the mfERG recordings from corresponding areas, the analysis indicated that there was a clear correlation between the strength of mfERG responses and the cone density measurements, i.e., lower amplitudes were associated with lower cone density measurements confirming the results of Choi et al. (13) using AO images. Granse et al. (38) have demonstrated that mfERG and multifocal visual evoked potentials may be more useful for examining remaining visual function in RP patients than visual field measurements. These findings are consistent with the unspecific remaining extent of the visual fields measured in this study.

It has been shown that when corrected for age, the preserved kinetic visual field is significantly larger in USH2 than in USH1 (37) and patients with USH1 have vastly reduced visual fields already at an early age. A direct comparison of these two cohorts of different sizes and different clinical phenotypes is a limitation of this analysis. However, the heterogeneity of the patients inside our cohorts was high. For example, the 60-year-old USH1 patient 2 still had a comparatively large visual field, preserved scotopic ERG and his retinal layering was more widely preserved than the one of patient 6 (USH2). Due to this heterogeneity, we believe that these ten patients could be compared within the context of their phenotypic expression.

In conclusion, we find that FI-AO combined with SD-OCT and FAF imaging are useful and sensitive clinical imaging methods for examining photoreceptor alterations due to RP, although there are clear limitations of AO techniques in the clinical routine. An accurate quantification of structural alterations in the retina is fundamental for monitoring the success of future therapeutical interventions (41).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Ethics Committee of the University of Tuebingen. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

MK: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. SK: Writing – review & editing. KrS: Conceptualization, Formal analysis, Writing – review & editing. FN: Conceptualization, Investigation, Writing – review & editing. KaS: Funding acquisition, Writing – review & editing. FK: Formal analysis, Investigation, Writing – original draft.