Two-year prospective natural history study of EYS-associated retinitis pigmentosa using adaptive optics: The KEYS Study

Two-year prospective natural history study of EYS-associated retinitis pigmentosa using adaptive optics: The KEYS Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Two-year prospective natural history study of EYS-associated retinitis pigmentosa using adaptive optics: The KEYS Study Hiraoka Masakazu, Maeda Akiko, Yamamoto Midori, Gofas-Salas Elena, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8198915/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract This study was conducted to evaluate the longitudinal morphological changes in cone density (CD) in patients with the eyes shut homolog ( EYS )-associated retinitis pigmentosa (RP) using adaptive optics (AO) fundus imaging and to assess its potential as a biomarker for disease progression. As a sub-analysis of the KEYS study, this prospective observational study was conducted at the Kobe Eye Center, involving 50 patients with EYS -RP; 27 eyes from 27 patients who were eligible for adaptive optics fundus imaging were included in this analysis. Ellipsoid zone (EZ) length was assessed using optical coherence tomography, and mean deviation (MD) values were obtained from static visual field. CD showed a significant reduction in all regions of interest as early as 6 months from baseline. In contrast, a significant decrease in EZ length was observed only at 24 months, while MD values did not exhibit significant changes throughout the observation period. AO fundus imaging demonstrated high sensitivity in detecting early structural changes in EYS -RP. These findings contribute to a deeper understanding of the natural history of the disease and suggest that CD measurement could serve as a valuable biomarker for monitoring disease progression and evaluating treatment efficacy. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Medical research adaptive optics ophthalmoscopy photoreceptor retinitis pigmentosa inherited retinal dystrophy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy (IRD), affecting approximately 1 in 4,000 individuals, with mutations in more than 70 genes identified as causative factors[ 1 ]. RP is characterized by photoreceptor cell death leading to progressive retinal degeneration, resulting in symptoms such as night blindness and visual field loss. EYS is one of the major causative genes for RP globally and is particularly prevalent within the Japanese population[ 2 – 11 ]. Currently, no established effective treatments are available for RP, although research efforts are actively focusing on potential interventions, including cell and gene therapies[ 12 – 15 ]. As research into therapeutic options advances, the need for reliable parameters to evaluate visual function in patients with RP becomes increasingly necessary for assessing therapeutic efficacy and optimizing treatment strategies; however, such parameters remain poorly established. Retinal degenerative diseases typically progress slowly over several years, requiring significant photoreceptor loss before reliable and statistically significant visual functional changes can be measured. Structural deterioration, such as a decline in cone photoreceptor density or an increase in cone spacing, is known to precede functional impairment in patients with RP, thus highlighting the urgent need for validated structural biomarkers. Adaptive optics (AO) technology has been utilized in retinal disease research since the 1990s. This technology has been extensively applied to investigate various retinal disorders[ 16 , 17 ]. The resolution of retinal images is generally constrained by optical aberrations within the eye, rendering photoreceptor cells, which typically range in size from 2 to 5 µm, invisible to conventional ophthalmic imaging techniques[ 18 ]. AO techniques compensate for these aberrations, enabling in vivo visualization of photoreceptor cells, thereby enhancing our understanding of disease mechanisms[ 19 , 20 ]. The AO device used in the present study predominantly images the outer segments of cones[ 21 ]. Longitudinal assessments of cone density (CD) in RP have been reported by Duncan et al., who used AO scanning laser ophthalmoscopy (AO-SLO) to track changes in CD over time. Their study demonstrated the utility of CD as a sensitive structural biomarker for monitoring disease progression and treatment response in RP[ 22 ]. The purpose of this study was to assess longitudinal changes in cone photoreceptor density in patients with RP and to investigate correlations between structural changes and other visual functions. This sub-analysis was based on the KEYS study (UMIN000057025), a prospective observational study conducted at the Kobe Eye Center involving 50 patients with EYS -RP. Among them, 27 eyes from 27 patients who were eligible for adaptive optics fundus imaging were included in this analysis. Additionally, parameters including best-corrected visual acuity (VA), static visual field assessments via the Humphrey Field Analyzer, ellipsoid zone (EZ) length measured via optical coherence tomography (OCT), and genetic mutations were assessed. Relationships between changes in CD and these visual function parameters were analyzed. To our knowledge, this represents the largest prospective AO-based fundus imaging study in patients with EYS -RP. The findings may contribute to a better understanding of the natural history of RP and highlight the potential utility of AO imaging as a more sensitive parameter for evaluating therapeutic efficacy, thereby assisting in treatment selection and optimization for patients with RP. Results Clinical characteristics The clinical characteristics of the study participants are summarized in Table 1 . A total of 27 eyes were included in the study and classified into three groups (Groups A, B, and C). The mean age of all patients was 44.0 ± 11.5 years, with a male-to-female ratio of 1:2. The mean age in each group was 46 years in Group A (with only one eye), 40.1 ± 10.3 years in Group B, and 49.1 ± 11.9 years in Group C, with no significant differences in age among the groups. The axial length (AL) of all patients was 24.6 ± 1.11 mm, with similar values across the groups (Group A: 23.9 mm, Group B: 24.5 ± 1.13 mm, Group C: 24.9 ± 1.13 mm). The mean VA (logMAR) was 0.05 ± 0.20, with minimal differences among the groups. The mean EZ width was 2053 ± 1576 µm overall, but Group A exhibited a narrower width of 829 µm than that in the other groups. The average mean deviation (MD) value from visual field testing was − 15.5 ± 7.84 dB, with no remarkable differences among the groups. Subgroup analysis by genetic group was not performed because Group A included only one participant (n = 1), and no significant difference in CD was observed between Groups B and C. Table 1 Baseline characteristics of study participants All Groups Group A Group B Group C Number of eyes 27 1 15 11 Age (in years) 44.0 ± 11.5 46 40.1 ± 10.3 49.1 ± 11.9 Sex (Male, Female) 9, 18 1, 0 2, 13 6, 5 Axial length (mm) 24.6 ± 1.11 23.9 24.5 ± 1.13 24.9 ± 1.13 LogMAR 0.05 ± 0.20 -0.08 0.06 ± 0.21 0.05 ± 0.19 EZ width (µm) 2053 ± 1576 829 2416 ± 1757 1668 ± 1273 Mean deviation (dB) -15.5 ± 7.84 -19.8 -16.5 ± 8.10 -13.6 ± 7.77 Patients were classified into three distinct subgroups as follows: Group A: Homozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2)z; 2; Group B: Heterozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2); and Group C: Other EYS mutations strongly suspected to be pathogenic. EZ, ellipsoid zone Changes in CD over 2 years CD was measured longitudinally at baseline and every 6 months over 2 years (i.e., at 5 time points) using montage images covering a 4° area from the fovea (Fig. 1 ). The foveal center was identified by combining OCT and infrared images. CD changes were analyzed using linear mixed-effects models (LMMs), revealing significant reductions in all regions of interest (ROIs) (Fig. 2 , Table 2 ). Table 2 Estimated intercepts, monthly slopes, and annual reduction rates in CD for each ROI using LMMs ROI Intercept (cell/mm 2 ) Slope (cell/mm 2 /month) Reduction rate (%/month) p_value 2N 11139 ± 749 -204 ± 28 -1.83 ± 0.28 < 0.001 2T 11015 ± 717 -178 ± 27 -1.62 ± 0.28 < 0.001 4N 9310 ± 558 -161 ± 25 -1.73 ± 0.29 < 0.001 4T 10197 ± 542 -192 ± 23 -1.88 ± 0.25 < 0.001 ROI, region of interest; CD, cone density; LMM, linear mixed effects model; 2N, 2° nasal; 4N, 4° nasal; 2T, 2° temporal; 4T, 4° temporal Estimated baseline CD, monthly decline, and reduction rates are as follows: 2° nasal (2N): 11,139 cells/mm², -203.8 cells/mm²/month, -2.01%/year (p = 2.35 × 10⁻ 10 ); 2° temporal (2T): 11,015 cells/mm², -178.1 cells/mm²/month, -1.77%/year (p = 5.17 × 10⁻ 9 ); 4° nasal (4N): 9,310 cells/mm², -161.1 cells/mm²/month, -1.89%/month (p = 9.39 × 10⁻ 9 ); 4° temporal (4T): 10,197 cells/mm², -192.4 cells/mm²/month, -2.1%/month (p = 8.79 × 10⁻ 13 ). Changes in other parameters over 2 years The longitudinal changes in logMAR VA, EZ width, and MD values are presented in (Table 3 ). A statistically significant decline in logMAR VA was first observed at 18 months (p = 0.0066). Regarding the horizontal EZ length, significant shortening was detected for the first time at 24 months (p = 0.00039); however, no significant reduction was observed in the vertical EZ length. In contrast, MD values from visual field testing did not exhibit any significant changes over the observation period. Table 3 Measured EZ width, MD, and VA over 24 months Baseline (at 0 months) At 6 months At 12 months At 18 months At 24 months EZ (µm) 2052.6 ± 1575.9 1981.4 ± 1521.0 1941.6 ± 1563.2 1912.0 ± 1542.8 1804.1 ± 1450.1 MD (dB) -15.5 ± 7.8 -15.8 ± 7.7 -15.7 ± 7.9 -15.8 ± 7.4 -15.7 ± 7.2 VA (logMAR) 0.05 ± 0.19 0.07 ± 0.18 0.1 ± 0.24 0.11 ± 0.22 0.12 ± 0.23 EZ, ellipsoid zone; MD, mean deviation; VA, visual acuity Correlation analysis A correlation analysis was performed to evaluate the relationship between CD and other parameters (Table 4 , Supplementary Fig. S1 ). As previously mentioned, the mean total deviation (TD)68 value and mean of the four foveal points were also examined for MD. The results indicated no significant correlation with any of the parameters analyzed (mean correlation coefficient with MD: r = -0.10, mean correlation coefficient with EZ width: r = -0.13). The correlation coefficient between C4 and CD had a mean value of -0.25, indicating a weak negative correlation overall. At month 6, a relatively stronger negative correlation was observed (r = -0.43), but at month 0 (-0.06) and month 18 (-0.12), the correlation was minimal. Similarly, the mean correlation coefficient between TD 68 and CD was − 0.17, showing a weak negative correlation, akin to C4. Notably, at month 6, the correlation coefficient was − 0.27, and at month 24, it was − 0.3, but no consistent trend was observed across time points. A regression analysis based on scatter plots was conducted to further examine these relationships. The regression slope between CD and MD was very small (slope = -25.4), with a coefficient of determination (R²) of 0.0088, indicating no significant association. The relationship between C4 and CD was also analyzed using scatter plots, yielding an R² value of 0.0309. While a weak negative trend was observed, no definitive correlation was established. The data distribution was broad, with considerable variation in CD values across both high and low C4 levels. Table 4 Pearson’s correlation analysis of CD with EZ, MD, C4, TD 68 , and VA in all patients Mean CD of four regions vs. Baseline (at 0 months) At 6 months At 12 months At 18 months At 24 months Average MD (dB) 0.26 -0.28 -0.11 0.002 -0.35 -0.1 C4 -0.06 -0.43 -0.27 -0.12 -0.37 -0.25 TD 68 -0.08 -0.27 -0.17 -0.008 -0.3 -0.17 EZ width (µm) -0.29 0.02 -0.1 -0.03 -0.22 -0.13 VA (logMAR) 0.12 0.24 0.05 0.12 0.6 0.23 CD, cone density; EZ, ellipsoid zone; MD, mean deviation; VA, visual acuity Similarly, no clear correlation was observed between TD 68 and CD, with an extremely low R² value, and an overall inconsistent data trend. Further analysis of the relationship between CD and logMAR VA revealed a regression slope of 23.6, with an R² value of 0.0129, indicating a weak positive correlation, although its significance was highly limited. Additionally, the regression slope between CD and EZ width was extremely small (slope = -0.17), and the R² value was 0.0067, confirming an absence of a meaningful correlation. Overall, these results suggest no significant linear relationship between CD and MD, EZ width, or logMAR VA. The lack of strong correlations indicates that changes in CD did not directly correspond to functional impairments. Discussion In this study, we prospectively investigated 27 patients with RP and included assessments incorporating VA, visual field testing, OCT, and AO imaging. We further analyzed longitudinal changes in these parameters and their interrelationships. The results demonstrated a statistically significant reduction in horizontal EZ width at the 24-month follow-up, whereas logMAR VA exhibited a significant decline at 18 months. In contrast, MD values remained stable without significant changes throughout the follow-up period. The longitudinal analysis of CD changes within each ROI revealed a progressive decline over time in all regions (2N, 4N, 2T, and 4T), with statistically significant reductions observed across all observation periods (p < 0.001). Previous studies using AO imaging in healthy individuals have reported CD of approximately 27,850–44,500 cells/mm² in the 2° eccentric region and 21,000–32,000 cells/mm² in the 4° region. In comparison, the mean CD values at baseline obtained in the present study were markedly reduced. These findings indicate a substantial reduction in CD among patients with EYS -RP compared with that in normal eyes[ 23 – 26 ]. The annual reduction in CD across all four ROIs was significantly greater than the reported decline in healthy individuals[ 23 ]. Furthermore, CD exhibited considerable variability among patients, with some presenting values below 5,000 cells/mm². These findings may reflect a characteristic of RP, in which diverse phenotypic expressions can occur even among individuals with the same causative gene[ 7 ]. Regionally, the 4° temporal region exhibited the greatest decline, which aligns with the characteristic pattern of RP, where photoreceptor degeneration typically progresses outward from the surrounding regions[ 27 ]. Correlation analysis revealed no clear associations between cone density and functional parameters. These findings suggest that cone density may decline independently of changes in visual function, indicating the potential value of structural biomarkers in assessing disease progression. The weak correlation between CD and MD suggests that overall visual field sensitivity decline does not directly correlate with cone density reduction. While MD remained relatively stable, CD showed an early and significant decline, indicating that CD loss may precede measurable functional impairment. Similarly, the limited correlation between CD and EZ width suggests that the relationship between EZ shortening and CD reduction may not adhere to a simple linear relationship. Likewise, no strong associations emerged between CD and either C4 or TD 68 , suggesting that visual field sensitivity may be influenced not only by localized alterations in CD but also by broader compensatory mechanisms within the retina or neural pathways, consistent with a previous report[ 28 ]. The findings our study indicate that CD changes may be detected earlier than functional measures in EYS -RP. While reductions in EZ width were significant at 24 months, CD showed significant changes much earlier, reinforcing its potential as a sensitive marker for disease progression. The lack of strong correlations between CD and functional parameters (MD, EZ width, VA) emphasizes the necessity for a comprehensive evaluation that integrates multiple indicators to accurately assess disease progression. By directly visualizing and quantifying CD using AO imaging, this study successfully captured 2-year disease progression in hereditary retinal degeneration with higher sensitivity than that of other functional tests. The findings suggest that CD measurement could serve as an objective and highly sensitive marker for treatment monitoring and therapeutic efficacy assessment. However, this study had certain limitations. First, the ROIs were placed along the horizontal meridian (nasal and temporal sides), and measurements in the vertical direction (superior and inferior) were not assessed. Therefore, data regarding vertical asymmetry are not available. Second, in patients with advanced retinal degeneration or older age, fixation instability made it difficult to obtain AO images or resulted in sub-optimal images. The imaging modality used in this study was AO flood-illuminated ophthalmoscopy, which simultaneously captures the entirety of the ROI using backscattered light. While this technique allows for shorter capture times, it is also more susceptible to intraocular light scattering and has lower resolution than that of AO-SLO[ 29 ]. In addition, the AO device used in this study was unable to accurately measure CD at the foveal center due to the high density of cones in this region. Future studies may overcome this limitation by incorporating vertical meridian measurements to evaluate superior-inferior asymmetry and by applying advanced AO imaging techniques such as Adaptive Optics Optical Coherence Tomography (AO-OCT), which enables more precise assessment by accounting for the three-dimensional architecture of the retina. Improvements in eye-tracking and image registration technology may also help overcome fixation instability in older patients or those with advanced retinal degeneration. Furthermore, the development of adaptive AO systems with enhanced axial resolution could enable reliable CD measurements even at the foveal center. In conclusion, in this study, we conducted a 2-year prospective investigation of patients with EYS -RP, contributing valuable insights into the natural history of the disease. Furthermore, AO imaging demonstrated higher sensitivity in assessing retinal structural changes than that of traditional ophthalmic examinations. Given its ability to detect early retinal changes and disease progression, AO imaging has the potential to serve as a valuable tool for optimizing treatment strategies and evaluating post-treatment outcomes. Methods Ethical guidelines Patient recruitment was conducted between March 1, 2021 and September 30, 2024. Before collecting the samples from the patients, written informed consent was obtained in accordance with the guidelines of the Declaration of Helsinki. Samples from all patients and their family members were collected accordingly. The study was approved by the Research Review Committee of Kobe City Kobe Eye Center Hospital (Permit No. : ek241201) and by the Institutional Review Board at the Research Committee of Santen Pharmaceutical Co., Ltd. The study was registered at the University Hospital Medical Information Network Clinical Trials Registry (clinical trial identifier: UMIN000057025). Genomic DNA was isolated from EDTA blood according to standard protocols. Data were obtained between March 1, 2021 and March 31, 2024. The authors did not have access to any personally identifiable information of the participants during and after data collection. Patients This study included 27 patients with EYS -RP who visited the Kobe City Eye Hospital. A comprehensive ophthalmological examination was performed for diagnostic purposes, confirming RP based on bilateral visual loss, night blindness, visual field constriction, narrow retinal vessels, coarse retinal pigmentation, bone spicule pigmentation, white spots, optic nerve atrophy, and macular degeneration observed in fundus examinations. Visual fields were examined using Humphrey's Field Analyzer (Carl Zeiss-Humphrey Systems, Dublin, California, USA) and Goldmann perimetry (Haag-Streit, Bern, Switzerland). Electroretinograms (LE-4000; Tomey, Nagoya, Japan) were examined for attenuation and loss. Retinal pigment epithelium and photoreceptor cells were also evaluated using fundus autofluorescence (Optos 200Tx; Optos, Dunfermline, Scotland) and OCT (Spectralis; Heidelberg Engineering, Heidelberg, Germany). Cone photoreceptors were imaged using an AO fundus camera (rtx1™, Imagine Eyes, Orsay, France). AL was measured using the IOL Master 700 (Carl Zeiss Meditec AG, Hennigsdorf, Germany). Examination protocol The inclusion criteria required that participants must be of any sex, aged 20 years or older, and diagnosed with RP caused by one of the following EYS mutations in both eyes. These patients were classified into three distinct subgroups as follows. 1. Group A: Homozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2) 2. Group B: Heterozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2) 3. Group C: Other EYS mutations strongly suspected to be pathogenic Additionally, at baseline measurement, at least one eye exhibited an MD value of -30 dB or greater as measured using the Humphrey Field Analyzer 10 − 2. To obtain high-quality AO images, only patients who could fixate on the target of the device were included. The study’s exclusion criteria included patients with best-corrected VA of less than 0.1 (indicating low vision) in both eyes. Additionally, patients diagnosed with glaucoma or ocular hypertension (intraocular pressure ≥ 22 mmHg) were excluded from the study. Patients with retinal lesions unrelated to RP in the study eye, such as retinal hemorrhage, retinal vascular occlusion, or proliferative diabetic retinopathy, were also excluded. Images exhibiting loss of focus, blink artifacts, or motion artifacts were excluded from this study. Next-generation sequencing (NGS) and variant analyses Targeted NGS using a 50-gene panel was performed for initial genetic testing (Supplementary Table S1 ). Targeted libraries were sequenced leveraging an Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). The detected variants were interpreted based on the criteria and guidelines recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[ 5 ]. The molecular diagnosis of each patient was reviewed by a multidisciplinary team that included ophthalmologists, clinical geneticists, optometrists, nurses, researchers, and genetic counselors. Multimodal image analysis/AO imaging This study utilized AO images obtained using an AO flood-illumination retinal camera (rtx1™, Imagine Eyes, France), the principles of which have been previously described in detail[ 30 ]. Image analysis was performed using the AOdetect mosaic software (Imagine Eyes, France), provided by the manufacturer. To account for the Stiles–Crawford effect of photoreceptors, we decided to obtain all AO images from the center of the pupil. AO images were captured to ensure adequate coverage of retinal areas up to 4° eccentricity from the fovea. The ROIs were selected at the eccentricities of 2° and 4° from the fovea (Fig. 1 ). The i2k Retina (Dural Align LLC, New York, USA) was employed to create a montage of AO images, with the objective of identifying the foveal center. This was achieved by superimposing the AO images with SLO and OCT images from Spectralis. Four ROIs were strategically positioned at 2° and 4° in the temporal and nasal directions, respectively. The ROI was obtained from superior and inferior points, and the CDs at these points were subsequently averaged (indicated by red squares in Fig. 1 ). For an AL of 24 mm, image size of 62 × 62 µm was determined[ 31 ]. We used the follow-up mode of AOdetect to analyze and measure the CD from identical ROIs. The ROIs were selected using the maximum density method, which has been described previously[ 25 , 26 ]. Measurements were corrected based on each participant’s AL. The software calculated local cell density (cells/mm²) and regularity (%) through the Voronoi analysis (Fig. 4 )[ 32 ]. Given that AO imaging may misidentify hyperreflective spots as cone photoreceptors, automated cone detection results were manually verified and corrected by two retinal specialists. OCT analysis OCT imaging was performed using the Spectralis device (Heidelberg Engineering, Heidelberg, Germany). EZ length was measured in both eyes using the "caliper" function of the internal software (Heidelberg Eye Explorer version 6.12.3.0), and the same OCT images used for AO analysis were employed for this measurement. The analysis involved horizontal OCT scans aligned with the ROIs defined by AO images. Three ophthalmologists performed the measurements, and the average of their readings constituted the final measurement. Humphrey Field Analyzer analysis All patients underwent automated static perimetry using the HF10-2 SITA standard program. Reliability criteria included a fixation loss score of ≤ 20%, with false-positive or false-negative rate set at ≤ 33%. Data were excluded from the study if repeat tests did not yield reliable results. Considering that dB values are logarithmically scaled, averaging dB sensitivity across points was deemed inappropriate[ 33 ]. Therefore, dB sensitivity values were first converted to 1/Lambert, averaged, and then transformed back into the dB scale as "antilogged first average (dB)" for analysis[ 33 ]. Each TD value was first antilogged and then averaged. To return to the dB scale, the values were log-transformed again, yielding the “antilogged first average (ALFA)” (dB) values. ALFA was analyzed for both the overall average (68 points were used for TD analysis [TD 68 ]) and the central four points (C4) (Fig. 1 ). Statistical analysis Comparisons between visits were conducted using paired t-tests with Bonferroni correction for multiple comparisons. To evaluate longitudinal changes in CD, LMMs were applied separately for each ROI to estimate the annual rate of decline and to assess whether VISIT had a statistically significant effect on CD. P-values for the fixed effects in the LMMs were calculated using Satterthwaite’s approximation. Associations between CD and other parameters (EZ, MD, VA, C4, and TD 68 ) were assessed using Pearson’s correlation coefficients. All measurements were independently evaluated by two ophthalmologists. For the correlation analyses, all available data points were included, even in instances where all five visits were not completed, provided the two variables of interest were measured at the same visit. Statistical analyses were performed using Microsoft Excel version 16.16.27 (Microsoft, Redmond, USA) and RStudio version 2024.04.2 + 764. Declarations Ethics declaration The study was approved by the Research Review Committee of Kobe City Kobe Eye Center Hospital (Permit No.: ek241201) and by the Institutional Review Board at the Research Committee of Santen Pharmaceutical Co., Ltd. The study was registered at the University Hospital Medical Information Network Clinical Trials Registry (clinical trial identifier: UMIN000057025). Competing interests A.M. received a financial support from Santen. Y.T. and M.T. are employees of Santen. H.M., M.Y., E.G., S.Y., S.K., Y.H., Y.K., and K.G. declare no competing interests. Funding Declaration This work was supported by Santen Pharmaceutical Co., Ltd. The funder had no role in study design, data collection, data analysis, or manuscript preparation. Consent to participate Before collecting the samples from the patients, written informed consent was obtained in accordance with the guidelines of the Declaration of Helsinki. Acknowledgements This work was supported by Santen. We thank the faculty and staff of Kobe City Eye Hospital and Kawasaki Medical School for their comments and discussion, the members of Imagine Eyes and Santen Pharmaceutical Co., Ltd. for their support, and Editage (www.editage.jp) for English language editing. Author contributions K.G. and A.M. designed and conducted the study. K.G., M.Y., E.G., S.K., S.Y., Y.T., and H.M. performed the data acquisition and analysis, and H.M. drafted the manuscript. M.T., Y.H., and Y.K. provided critical input during manuscript preparation. All authors reviewed and approved the final manuscript. Data availability statement The genetic variant data generated in this study have been deposited in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) under the accession numbers SCV007122111 to SCV007122125. All other data supporting the findings of this study are included within the article and its Supplementary Information files. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Supplementary figures and tables associated with the main findings are provided in the Supplementary Information file. References Consugar, M. B. et al. Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing. Genet. Med. 17 , 253–261. 10.1038/gim.2014.172 (2015). Pieras, J. I. et al. Copy-number variations in EYS: a significant event in the appearance of arRP. Invest. Ophthalmol. Vis. 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Longitudinal study of cone photoreceptors during retinal degeneration and in response to ciliary neurotrophic factor treatment. Invest. Ophthalmol. Vis. Sci. 52 , 2219–2226. 10.1167/iovs.10-6479 (2011). Song, H., Chui, T. Y., Zhong, Z., Elsner, A. E. & Burns, S. A. Variation of cone photoreceptor packing density with retinal eccentricity and age. Invest. Ophthalmol. Vis. Sci. 52 , 7376–7384. 10.1167/iovs.11-7199 (2011). Chui, T. Y., Song, H. & Burns, S. A. Adaptive-optics imaging of human cone photoreceptor distribution. J. Opt. Soc. Am. Opt. Image Sci. Vis. 25 , 3021–3029. 10.1364/josaa.25.003021 (2008). Gocho, K. et al. High-Resolution Adaptive Optics Retinal Image Analysis at Early Stage Central Areolar Choroidal Dystrophy With PRPH2 Mutation. Ophthalmic Surg. Lasers Imaging Retina . 47 , 1115–1126. 10.3928/23258160-20161130-05 (2016). Feng, S. et al. Assessment of Different Sampling Methods for Measuring and Representing Macular Cone Density Using Flood-Illuminated Adaptive Optics. Invest. Ophthalmol. Vis. Sci. 56 , 5751–5763. 10.1167/iovs.15-16954 (2015). Milam, A. H., Li, Z. Y. & Fariss, R. N. Histopathology of the human retina in retinitis pigmentosa. Prog Retin Eye Res. 17 , 175–205. 10.1016/s1350-9462(97)00012-8 (1998). Nakatake, S. et al. Early detection of cone photoreceptor cell loss in retinitis pigmentosa using adaptive optics scanning laser ophthalmoscopy. Graefes Arch. Clin. Exp. Ophthalmol. 257 , 1169–1181. 10.1007/s00417-019-04307-0 (2019). Ashourizadeh, H. et al. Pearls and Pitfalls of Adaptive Optics Ophthalmoscopy in Inherited Retinal Diseases. Diagnostics (Basel) . 13. 10.3390/diagnostics13142413 (2023). Ro-Mase, T. et al. Association Between Alterations of the Choriocapillaris Microcirculation and Visual Function and Cone Photoreceptors in Patients With Diabetes. Invest. Ophthalmol. Vis. Sci. 61 , 1. 10.1167/iovs.61.6.1 (2020). Zaleska-Zmijewska, A., Wawrzyniak, Z., Kupis, M. & Szaflik, J. P. The Relation between Body Mass Index and Retinal Photoreceptor Morphology and Microvascular Changes Measured with Adaptive Optics (rtx1) High-Resolution Imaging. J Ophthalmol 6642059, (2021). 10.1155/2021/6642059 (2021). Querques, G. et al. Adaptive Optics Imaging of Foveal Sparing in Geographic Atrophy Secondary to Age-Related Macular Degeneration. Retina 36 , 247–254. 10.1097/IAE.0000000000000692 (2016). Sayo, A. et al. Longitudinal study of visual field changes determined by Humphrey Field Analyzer 10 – 2 in patients with Retinitis Pigmentosa. Sci. Rep. 7 , 16383. 10.1038/s41598-017-16640-7 (2017). Additional Declarations Competing interest reported. A.M. received a financial support from Santen. Y.T. and M.T. are employees of Santen. H.M., M.Y., E.G., S.Y., S.K., Y.H., Y.K., and K.G. declare no competing interests. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 21 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviews received at journal 08 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers invited by journal 25 Feb, 2026 Editor invited by journal 24 Feb, 2026 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 21 Dec, 2025 First submitted to journal 21 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8198915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":597151429,"identity":"d3771de5-ba5c-43b1-8f69-f6ae7f149b6d","order_by":0,"name":"Hiraoka Masakazu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3QMQrCMBTG8U8C7RLpahDsFVIcpItnqQg6eQIHhUJX14qX0M2xErDLQ9eOFi+Qzg5qETetdhPJf0iy/OC9ACbTDyZZeSbogJXXs9YXpFuD4EEG868H69nNXGvqj9epnSfFFq6zUgn87Xvih3ZXxNlwslZc7pYELz6MAgiqGExZaHPNJsuQQzWjawDiEiKqJOzC9WwsQvt0JwhccvQnYrV5pgKH3d8lkcTxkfgxpd6ClbtE8DZkyaRyl+OeZXo/dS0nPesigtshlp9FxY+9Tol5XdIoahOTyWT6424vGk5WX5pVywAAAABJRU5ErkJggg==","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hiraoka","middleName":"","lastName":"Masakazu","suffix":""},{"id":597151433,"identity":"4f49af3a-686d-4e55-98de-ac3176fcbc52","order_by":1,"name":"Maeda Akiko","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Maeda","middleName":"","lastName":"Akiko","suffix":""},{"id":597151437,"identity":"3d2c7e26-aa3b-4101-9936-b3a6bdb9c5a1","order_by":2,"name":"Yamamoto Midori","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yamamoto","middleName":"","lastName":"Midori","suffix":""},{"id":597151442,"identity":"abd917d1-c7c8-4bfc-892c-6361a84c0cd8","order_by":3,"name":"Gofas-Salas Elena","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gofas-Salas","middleName":"","lastName":"Elena","suffix":""},{"id":597151444,"identity":"e4b5bd8f-a215-44a6-b1ab-f9f2cc8dc9ea","order_by":4,"name":"Kitahata Shohei","email":"","orcid":"","institution":"Yokohama City University","correspondingAuthor":false,"prefix":"","firstName":"Kitahata","middleName":"","lastName":"Shohei","suffix":""},{"id":597151446,"identity":"46a9acf4-4f87-4564-a2ff-8495b16ffc98","order_by":5,"name":"Yokota Satoshi","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yokota","middleName":"","lastName":"Satoshi","suffix":""},{"id":597151456,"identity":"2fb869a8-d784-4acc-a47f-7058595e3402","order_by":6,"name":"Togashi Yuki","email":"","orcid":"","institution":"Santen Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Togashi","middleName":"","lastName":"Yuki","suffix":""},{"id":597151458,"identity":"b6109d9c-e959-4624-b280-313d10e08489","order_by":7,"name":"Toshimori Masanao","email":"","orcid":"","institution":"Santen Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Toshimori","middleName":"","lastName":"Masanao","suffix":""},{"id":597151463,"identity":"9e5b2258-aa46-4c23-b34a-6176a0e802a0","order_by":8,"name":"Hirami Yasuhiko","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hirami","middleName":"","lastName":"Yasuhiko","suffix":""},{"id":597151466,"identity":"540dedf0-7478-447a-81b5-fe1f435ebe64","order_by":9,"name":"Kurimoto Yasuo","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kurimoto","middleName":"","lastName":"Yasuo","suffix":""},{"id":597151470,"identity":"8d13d922-06d2-4b0b-8463-75b6dff53a2a","order_by":10,"name":"Gocho Kiyoko","email":"","orcid":"","institution":"Kobe City Eye Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gocho","middleName":"","lastName":"Kiyoko","suffix":""}],"badges":[],"createdAt":"2025-11-25 05:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8198915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8198915/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103733372,"identity":"6e907fe9-f05a-4223-9714-5fc96639bf43","added_by":"auto","created_at":"2026-03-02 09:28:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8331039,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement areas and definitions of CD, EZ length, and visual field sensitivity\u003cbr\u003e\n(\u003cstrong\u003ea\u003c/strong\u003e) A montage of AO images was created to include regions at 2° and 4° eccentricities from the fovea. Four ROIs were defined: two in the temporal retina and two in the nasal retina. (\u003cstrong\u003eb\u003c/strong\u003e) EZ length was measured using OCT images corresponding to the same retinal area. (\u003cstrong\u003ec\u003c/strong\u003e) Test point definitions for the HFA 10-2 visual field program. The left panel displays all test locations. The middle panel highlights TD\u003csub\u003e68\u003c/sub\u003e, the 68 points used for total deviation (TD) analysis. The right panel shows the selected locations defined as C4, which correspond to the central four points relevant to CD correlation.\u003c/p\u003e\n\u003cp\u003eAO, adaptive optics; CD, cone density; EZ, ellipsoid zone; OCT, optical coherence tomography; ROI, region of interest; HFA, Humphrey Field Analyzer; TD, total deviation\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/2c40a4f07d6c030eedfc07c7.png"},{"id":103732976,"identity":"e512ef10-e7e6-48fa-92b4-1fcef412b6b9","added_by":"auto","created_at":"2026-03-02 09:26:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1331699,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-year changes in CD across each ROI in all patients\u003cbr\u003e\nLongitudinal changes in CD were evaluated using AO over a 2-year period in patients with \u003cem\u003eEYS\u003c/em\u003e-RP. CD was measured at 6-month intervals for each ROI: (\u003cstrong\u003ea\u003c/strong\u003e) 2° nasal (2N), (\u003cstrong\u003eb\u003c/strong\u003e) 4° nasal (4N), (\u003cstrong\u003ec\u003c/strong\u003e) 2° temporal (2T), and (\u003cstrong\u003ed\u003c/strong\u003e) 4° temporal (4T). Box plots represent the distribution of CD at each time point. Statistically significant changes are indicated (*p \u0026lt; 0.05, **p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eAO, adaptive optics; CD, cone density; ROI, region of interest; \u003cem\u003eEYS\u003c/em\u003e-RP, eyes shut homolog-related retinitis pigmentosa\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/5bf07b6c96210215df6851ce.png"},{"id":103733152,"identity":"54ae1953-bf07-41c2-b9be-2834715a5de4","added_by":"auto","created_at":"2026-03-02 09:27:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":621646,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-year changes in VA, EZ, and MD in all patients\u003cbr\u003e\nLongitudinal changes in VA, EZ, and MD were assessed every 6 months over a 2-year period. Box plots show the distribution at each time point, with statistically significant differences indicated (*p \u0026lt; 0.05, **p \u0026lt; 0.001). (\u003cstrong\u003ea\u003c/strong\u003e) VA showed a significant decline at M18. (\u003cstrong\u003eb\u003c/strong\u003e) EZ length demonstrated a significant reduction at M24. (\u003cstrong\u003ec\u003c/strong\u003e) MD did not show significant changes throughout the observation period.\u003c/p\u003e\n\u003cp\u003eVA, visual acuity; EZ, ellipsoid zone; MD, mean deviation; M18, month 18; M24, month 24\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/9ed778ab57c24802709f75ab.png"},{"id":103732890,"identity":"4ad82a2a-7cb5-43a7-a10d-90137c9b30d2","added_by":"auto","created_at":"2026-03-02 09:26:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10324175,"visible":true,"origin":"","legend":"\u003cp\u003eLongitudinal changes in CD in a representative case based on AO images\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) A montage AO image showing the ROI located 4° temporal to the fovea. CD was analyzed within this ROI. (\u003cstrong\u003eb\u003c/strong\u003e) Enlarged view of the white square in (\u003cstrong\u003ea\u003c/strong\u003e). CD was measured within the yellow square area. (\u003cstrong\u003ec\u003c/strong\u003e) Longitudinal changes in CD in the same ROI at baseline (M0), 6 months (M6), 12 months (M12), 18 months (M18), and 24 months (M24). Scale bars indicate 100 µm.\u003c/p\u003e\n\u003cp\u003eAO, adaptive optics; CD, cone density; ROI, region of interest\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/0639586b07b8d4aabeb91963.png"},{"id":103733762,"identity":"397cf066-b2ae-4ec3-9992-0ce541581b5b","added_by":"auto","created_at":"2026-03-02 09:29:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19601207,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/d7f40e61-29e5-400b-a54c-d6cf56ea6fb0.pdf"},{"id":103732919,"identity":"6cae7052-f1d5-4a71-9e32-5021734315ed","added_by":"auto","created_at":"2026-03-02 09:26:41","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":432633,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8198915/v1/85ab79332e34821ff9a3eee1.pdf"}],"financialInterests":"Competing interest reported. A.M. received a financial support from Santen. Y.T. and M.T. are employees of Santen. H.M., M.Y., E.G., S.Y., S.K., Y.H., Y.K., and K.G. declare no competing interests.","formattedTitle":"Two-year prospective natural history study of EYS-associated retinitis pigmentosa using adaptive optics: The KEYS Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRetinitis pigmentosa (RP) is the most common inherited retinal dystrophy (IRD), affecting approximately 1 in 4,000 individuals, with mutations in more than 70 genes identified as causative factors[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. RP is characterized by photoreceptor cell death leading to progressive retinal degeneration, resulting in symptoms such as night blindness and visual field loss. \u003cem\u003eEYS\u003c/em\u003e is one of the major causative genes for RP globally and is particularly prevalent within the Japanese population[\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Currently, no established effective treatments are available for RP, although research efforts are actively focusing on potential interventions, including cell and gene therapies[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs research into therapeutic options advances, the need for reliable parameters to evaluate visual function in patients with RP becomes increasingly necessary for assessing therapeutic efficacy and optimizing treatment strategies; however, such parameters remain poorly established. Retinal degenerative diseases typically progress slowly over several years, requiring significant photoreceptor loss before reliable and statistically significant visual functional changes can be measured. Structural deterioration, such as a decline in cone photoreceptor density or an increase in cone spacing, is known to precede functional impairment in patients with RP, thus highlighting the urgent need for validated structural biomarkers.\u003c/p\u003e \u003cp\u003eAdaptive optics (AO) technology has been utilized in retinal disease research since the 1990s. This technology has been extensively applied to investigate various retinal disorders[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe resolution of retinal images is generally constrained by optical aberrations within the eye, rendering photoreceptor cells, which typically range in size from 2 to 5 \u0026micro;m, invisible to conventional ophthalmic imaging techniques[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. AO techniques compensate for these aberrations, enabling in vivo visualization of photoreceptor cells, thereby enhancing our understanding of disease mechanisms[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The AO device used in the present study predominantly images the outer segments of cones[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Longitudinal assessments of cone density (CD) in RP have been reported by Duncan et al., who used AO scanning laser ophthalmoscopy (AO-SLO) to track changes in CD over time. Their study demonstrated the utility of CD as a sensitive structural biomarker for monitoring disease progression and treatment response in RP[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe purpose of this study was to assess longitudinal changes in cone photoreceptor density in patients with RP and to investigate correlations between structural changes and other visual functions. This sub-analysis was based on the KEYS study (UMIN000057025), a prospective observational study conducted at the Kobe Eye Center involving 50 patients with \u003cem\u003eEYS\u003c/em\u003e-RP. Among them, 27 eyes from 27 patients who were eligible for adaptive optics fundus imaging were included in this analysis. Additionally, parameters including best-corrected visual acuity (VA), static visual field assessments via the Humphrey Field Analyzer, ellipsoid zone (EZ) length measured via optical coherence tomography (OCT), and genetic mutations were assessed. Relationships between changes in CD and these visual function parameters were analyzed. To our knowledge, this represents the largest prospective AO-based fundus imaging study in patients with \u003cem\u003eEYS\u003c/em\u003e-RP. The findings may contribute to a better understanding of the natural history of RP and highlight the potential utility of AO imaging as a more sensitive parameter for evaluating therapeutic efficacy, thereby assisting in treatment selection and optimization for patients with RP.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics\u003c/h2\u003e \u003cp\u003eThe clinical characteristics of the study participants are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 27 eyes were included in the study and classified into three groups (Groups A, B, and C). The mean age of all patients was 44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5 years, with a male-to-female ratio of 1:2. The mean age in each group was 46 years in Group A (with only one eye), 40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 years in Group B, and 49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9 years in Group C, with no significant differences in age among the groups. The axial length (AL) of all patients was 24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 mm, with similar values across the groups (Group A: 23.9 mm, Group B: 24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13 mm, Group C: 24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13 mm). The mean VA (logMAR) was 0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, with minimal differences among the groups. The mean EZ width was 2053\u0026thinsp;\u0026plusmn;\u0026thinsp;1576 \u0026micro;m overall, but Group A exhibited a narrower width of 829 \u0026micro;m than that in the other groups. The average mean deviation (MD) value from visual field testing was \u0026minus;\u0026thinsp;15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.84 dB, with no remarkable differences among the groups. Subgroup analysis by genetic group was not performed because Group A included only one participant (n\u0026thinsp;=\u0026thinsp;1), and no significant difference in CD was observed between Groups B and C.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAll Groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (in years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (Male, Female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9, 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1, 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2, 13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6, 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAxial length (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLogMAR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEZ width (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2053\u0026thinsp;\u0026plusmn;\u0026thinsp;1576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e829\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2416\u0026thinsp;\u0026plusmn;\u0026thinsp;1757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1668\u0026thinsp;\u0026plusmn;\u0026thinsp;1273\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean deviation (dB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-19.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003ePatients were classified into three distinct subgroups as follows: Group A: Homozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2)z; 2; Group B: Heterozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2); and Group C: Other \u003cem\u003eEYS\u003c/em\u003e mutations strongly suspected to be pathogenic.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eEZ, ellipsoid zone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChanges in CD over 2 years\u003c/h3\u003e\n\u003cp\u003eCD was measured longitudinally at baseline and every 6 months over 2 years (i.e., at 5 time points) using montage images covering a 4\u0026deg; area from the fovea (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The foveal center was identified by combining OCT and infrared images. CD changes were analyzed using linear mixed-effects models (LMMs), revealing significant reductions in all regions of interest (ROIs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated intercepts, monthly slopes, and annual reduction rates in CD for each ROI using LMMs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eROI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntercept (cell/mm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlope (cell/mm\u003csup\u003e2\u003c/sup\u003e/month)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReduction rate (%/month)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep_value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11139\u0026thinsp;\u0026plusmn;\u0026thinsp;749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-204\u0026thinsp;\u0026plusmn;\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11015\u0026thinsp;\u0026plusmn;\u0026thinsp;717\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-178\u0026thinsp;\u0026plusmn;\u0026thinsp;27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9310\u0026thinsp;\u0026plusmn;\u0026thinsp;558\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-161\u0026thinsp;\u0026plusmn;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10197\u0026thinsp;\u0026plusmn;\u0026thinsp;542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-192\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eROI, region of interest; CD, cone density; LMM, linear mixed effects model; 2N, 2\u0026deg; nasal; 4N, 4\u0026deg; nasal; 2T, 2\u0026deg; temporal; 4T, 4\u0026deg; temporal\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEstimated baseline CD, monthly decline, and reduction rates are as follows: 2\u0026deg; nasal (2N): 11,139 cells/mm\u0026sup2;, -203.8 cells/mm\u0026sup2;/month, -2.01%/year (p\u0026thinsp;=\u0026thinsp;2.35 \u0026times; 10⁻\u003csup\u003e10\u003c/sup\u003e); 2\u0026deg; temporal (2T): 11,015 cells/mm\u0026sup2;, -178.1 cells/mm\u0026sup2;/month, -1.77%/year (p\u0026thinsp;=\u0026thinsp;5.17 \u0026times; 10⁻\u003csup\u003e9\u003c/sup\u003e); 4\u0026deg; nasal (4N): 9,310 cells/mm\u0026sup2;, -161.1 cells/mm\u0026sup2;/month, -1.89%/month (p\u0026thinsp;=\u0026thinsp;9.39 \u0026times; 10⁻\u003csup\u003e9\u003c/sup\u003e); 4\u0026deg; temporal (4T): 10,197 cells/mm\u0026sup2;, -192.4 cells/mm\u0026sup2;/month, -2.1%/month (p\u0026thinsp;=\u0026thinsp;8.79 \u0026times; 10⁻\u003csup\u003e13\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eChanges in other parameters over 2 years\u003c/h3\u003e\n\u003cp\u003eThe longitudinal changes in logMAR VA, EZ width, and MD values are presented in (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A statistically significant decline in logMAR VA was first observed at 18 months (p\u0026thinsp;=\u0026thinsp;0.0066). Regarding the horizontal EZ length, significant shortening was detected for the first time at 24 months (p\u0026thinsp;=\u0026thinsp;0.00039); however, no significant reduction was observed in the vertical EZ length. In contrast, MD values from visual field testing did not exhibit any significant changes over the observation period.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasured EZ width, MD, and VA over 24 months\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaseline (at 0 months)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAt 6 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAt 12 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAt 18 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAt 24 months\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEZ (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2052.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1575.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1981.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1521.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1941.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1563.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1912.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1542.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1804.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1450.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMD (dB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-15.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e-15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVA (logMAR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eEZ, ellipsoid zone; MD, mean deviation; VA, visual acuity\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCorrelation analysis\u003c/h3\u003e\n\u003cp\u003eA correlation analysis was performed to evaluate the relationship between CD and other parameters (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As previously mentioned, the mean total deviation (TD)68 value and mean of the four foveal points were also examined for MD. The results indicated no significant correlation with any of the parameters analyzed (mean correlation coefficient with MD: r = -0.10, mean correlation coefficient with EZ width: r = -0.13). The correlation coefficient between C4 and CD had a mean value of -0.25, indicating a weak negative correlation overall. At month 6, a relatively stronger negative correlation was observed (r = -0.43), but at month 0 (-0.06) and month 18 (-0.12), the correlation was minimal. Similarly, the mean correlation coefficient between TD\u003csub\u003e68\u003c/sub\u003e and CD was \u0026minus;\u0026thinsp;0.17, showing a weak negative correlation, akin to C4. Notably, at month 6, the correlation coefficient was \u0026minus;\u0026thinsp;0.27, and at month 24, it was \u0026minus;\u0026thinsp;0.3, but no consistent trend was observed across time points. A regression analysis based on scatter plots was conducted to further examine these relationships. The regression slope between CD and MD was very small (slope = -25.4), with a coefficient of determination (R\u0026sup2;) of 0.0088, indicating no significant association. The relationship between C4 and CD was also analyzed using scatter plots, yielding an R\u0026sup2; value of 0.0309. While a weak negative trend was observed, no definitive correlation was established. The data distribution was broad, with considerable variation in CD values across both high and low C4 levels.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePearson\u0026rsquo;s correlation analysis of CD with EZ, MD, C4, TD\u003csub\u003e68\u003c/sub\u003e, and VA in all patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean CD of four regions vs.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaseline (at 0 months)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAt 6 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAt 12 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAt 18 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAt 24 months\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMD (dB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTD\u003csub\u003e68\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEZ width (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVA (logMAR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eCD, cone density; EZ, ellipsoid zone; MD, mean deviation; VA, visual acuity\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilarly, no clear correlation was observed between TD\u003csub\u003e68\u003c/sub\u003e and CD, with an extremely low R\u0026sup2; value, and an overall inconsistent data trend. Further analysis of the relationship between CD and logMAR VA revealed a regression slope of 23.6, with an R\u0026sup2; value of 0.0129, indicating a weak positive correlation, although its significance was highly limited. Additionally, the regression slope between CD and EZ width was extremely small (slope = -0.17), and the R\u0026sup2; value was 0.0067, confirming an absence of a meaningful correlation. Overall, these results suggest no significant linear relationship between CD and MD, EZ width, or logMAR VA. The lack of strong correlations indicates that changes in CD did not directly correspond to functional impairments.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we prospectively investigated 27 patients with RP and included assessments incorporating VA, visual field testing, OCT, and AO imaging. We further analyzed longitudinal changes in these parameters and their interrelationships. The results demonstrated a statistically significant reduction in horizontal EZ width at the 24-month follow-up, whereas logMAR VA exhibited a significant decline at 18 months. In contrast, MD values remained stable without significant changes throughout the follow-up period. The longitudinal analysis of CD changes within each ROI revealed a progressive decline over time in all regions (2N, 4N, 2T, and 4T), with statistically significant reductions observed across all observation periods (p \u0026lt; 0.001).\u003c/p\u003e \u003cp\u003ePrevious studies using AO imaging in healthy individuals have reported CD of approximately 27,850–44,500 cells/mm² in the 2° eccentric region and 21,000–32,000 cells/mm² in the 4° region. In comparison, the mean CD values at baseline obtained in the present study were markedly reduced. These findings indicate a substantial reduction in CD among patients with \u003cem\u003eEYS\u003c/em\u003e-RP compared with that in normal eyes[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. The annual reduction in CD across all four ROIs was significantly greater than the reported decline in healthy individuals[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, CD exhibited considerable variability among patients, with some presenting values below 5,000 cells/mm². These findings may reflect a characteristic of RP, in which diverse phenotypic expressions can occur even among individuals with the same causative gene[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Regionally, the 4° temporal region exhibited the greatest decline, which aligns with the characteristic pattern of RP, where photoreceptor degeneration typically progresses outward from the surrounding regions[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCorrelation analysis revealed no clear associations between cone density and functional parameters. These findings suggest that cone density may decline independently of changes in visual function, indicating the potential value of structural biomarkers in assessing disease progression.\u003c/p\u003e \u003cp\u003eThe weak correlation between CD and MD suggests that overall visual field sensitivity decline does not directly correlate with cone density reduction. While MD remained relatively stable, CD showed an early and significant decline, indicating that CD loss may precede measurable functional impairment. Similarly, the limited correlation between CD and EZ width suggests that the relationship between EZ shortening and CD reduction may not adhere to a simple linear relationship. Likewise, no strong associations emerged between CD and either C4 or TD\u003csub\u003e68\u003c/sub\u003e, suggesting that visual field sensitivity may be influenced not only by localized alterations in CD but also by broader compensatory mechanisms within the retina or neural pathways, consistent with a previous report[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings our study indicate that CD changes may be detected earlier than functional measures in \u003cem\u003eEYS\u003c/em\u003e-RP. While reductions in EZ width were significant at 24 months, CD showed significant changes much earlier, reinforcing its potential as a sensitive marker for disease progression. The lack of strong correlations between CD and functional parameters (MD, EZ width, VA) emphasizes the necessity for a comprehensive evaluation that integrates multiple indicators to accurately assess disease progression.\u003c/p\u003e \u003cp\u003eBy directly visualizing and quantifying CD using AO imaging, this study successfully captured 2-year disease progression in hereditary retinal degeneration with higher sensitivity than that of other functional tests. The findings suggest that CD measurement could serve as an objective and highly sensitive marker for treatment monitoring and therapeutic efficacy assessment.\u003c/p\u003e \u003cp\u003eHowever, this study had certain limitations. First, the ROIs were placed along the horizontal meridian (nasal and temporal sides), and measurements in the vertical direction (superior and inferior) were not assessed. Therefore, data regarding vertical asymmetry are not available. Second, in patients with advanced retinal degeneration or older age, fixation instability made it difficult to obtain AO images or resulted in sub-optimal images. The imaging modality used in this study was AO flood-illuminated ophthalmoscopy, which simultaneously captures the entirety of the ROI using backscattered light. While this technique allows for shorter capture times, it is also more susceptible to intraocular light scattering and has lower resolution than that of AO-SLO[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, the AO device used in this study was unable to accurately measure CD at the foveal center due to the high density of cones in this region. Future studies may overcome this limitation by incorporating vertical meridian measurements to evaluate superior-inferior asymmetry and by applying advanced AO imaging techniques such as Adaptive Optics Optical Coherence Tomography (AO-OCT), which enables more precise assessment by accounting for the three-dimensional architecture of the retina. Improvements in eye-tracking and image registration technology may also help overcome fixation instability in older patients or those with advanced retinal degeneration. Furthermore, the development of adaptive AO systems with enhanced axial resolution could enable reliable CD measurements even at the foveal center.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study, we conducted a 2-year prospective investigation of patients with \u003cem\u003eEYS\u003c/em\u003e-RP, contributing valuable insights into the natural history of the disease. Furthermore, AO imaging demonstrated higher sensitivity in assessing retinal structural changes than that of traditional ophthalmic examinations.\u003c/p\u003e \u003cp\u003eGiven its ability to detect early retinal changes and disease progression, AO imaging has the potential to serve as a valuable tool for optimizing treatment strategies and evaluating post-treatment outcomes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eEthical guidelines\u003c/h2\u003e\u003cp\u003ePatient recruitment was conducted between March 1, 2021 and September 30, 2024. Before collecting the samples from the patients, written informed consent was obtained in accordance with the guidelines of the Declaration of Helsinki. Samples from all patients and their family members were collected accordingly. The study was approved by the Research Review Committee of Kobe City Kobe Eye Center Hospital (Permit No. : ek241201) and by the Institutional Review Board at the Research Committee of Santen Pharmaceutical Co., Ltd. The study was registered at the University Hospital Medical Information Network Clinical Trials Registry (clinical trial identifier: UMIN000057025). Genomic DNA was isolated from EDTA blood according to standard protocols. Data were obtained between March 1, 2021 and March 31, 2024. The authors did not have access to any personally identifiable information of the participants during and after data collection.\u003c/p\u003e\n\u003ch3\u003ePatients\u003c/h3\u003e\n\u003cp\u003eThis study included 27 patients with \u003cem\u003eEYS\u003c/em\u003e-RP who visited the Kobe City Eye Hospital. A comprehensive ophthalmological examination was performed for diagnostic purposes, confirming RP based on bilateral visual loss, night blindness, visual field constriction, narrow retinal vessels, coarse retinal pigmentation, bone spicule pigmentation, white spots, optic nerve atrophy, and macular degeneration observed in fundus examinations. Visual fields were examined using Humphrey's Field Analyzer (Carl Zeiss-Humphrey Systems, Dublin, California, USA) and Goldmann perimetry (Haag-Streit, Bern, Switzerland). Electroretinograms (LE-4000; Tomey, Nagoya, Japan) were examined for attenuation and loss. Retinal pigment epithelium and photoreceptor cells were also evaluated using fundus autofluorescence (Optos 200Tx; Optos, Dunfermline, Scotland) and OCT (Spectralis; Heidelberg Engineering, Heidelberg, Germany). Cone photoreceptors were imaged using an AO fundus camera (rtx1\u0026trade;, Imagine Eyes, Orsay, France). AL was measured using the IOL Master 700 (Carl Zeiss Meditec AG, Hennigsdorf, Germany).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExamination protocol\u003c/h2\u003e \u003cp\u003eThe inclusion criteria required that participants must be of any sex, aged 20 years or older, and diagnosed with RP caused by one of the following \u003cem\u003eEYS\u003c/em\u003e mutations in both eyes. These patients were classified into three distinct subgroups as follows.\u003c/p\u003e\u003cp\u003e\u003cspan\u003e1. Group A: Homozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2)\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e2. Group B: Heterozygous for Exon26:c.4957dupA(Ser 1653 Lys fs*2)\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e3. Group C: Other\u0026nbsp;\u003cem\u003eEYS\u003c/em\u003e mutations strongly suspected to be pathogenic\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eAdditionally, at baseline measurement, at least one eye exhibited an MD value of -30 dB or greater as measured using the Humphrey Field Analyzer 10\u0026thinsp;\u0026minus;\u0026thinsp;2. To obtain high-quality AO images, only patients who could fixate on the target of the device were included.\u003c/p\u003e \u003cp\u003eThe study\u0026rsquo;s exclusion criteria included patients with best-corrected VA of less than 0.1 (indicating low vision) in both eyes. Additionally, patients diagnosed with glaucoma or ocular hypertension (intraocular pressure\u0026thinsp;\u0026ge;\u0026thinsp;22 mmHg) were excluded from the study. Patients with retinal lesions unrelated to RP in the study eye, such as retinal hemorrhage, retinal vascular occlusion, or proliferative diabetic retinopathy, were also excluded. Images exhibiting loss of focus, blink artifacts, or motion artifacts were excluded from this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNext-generation sequencing (NGS) and variant analyses\u003c/h2\u003e \u003cp\u003eTargeted NGS using a 50-gene panel was performed for initial genetic testing (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Targeted libraries were sequenced leveraging an Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). The detected variants were interpreted based on the criteria and guidelines recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The molecular diagnosis of each patient was reviewed by a multidisciplinary team that included ophthalmologists, clinical geneticists, optometrists, nurses, researchers, and genetic counselors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMultimodal image analysis/AO imaging\u003c/h2\u003e \u003cp\u003eThis study utilized AO images obtained using an AO flood-illumination retinal camera (rtx1\u0026trade;, Imagine Eyes, France), the principles of which have been previously described in detail[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Image analysis was performed using the AOdetect mosaic software (Imagine Eyes, France), provided by the manufacturer.\u003c/p\u003e \u003cp\u003eTo account for the Stiles\u0026ndash;Crawford effect of photoreceptors, we decided to obtain all AO images from the center of the pupil. AO images were captured to ensure adequate coverage of retinal areas up to 4\u0026deg; eccentricity from the fovea. The ROIs were selected at the eccentricities of 2\u0026deg; and 4\u0026deg; from the fovea (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe i2k Retina (Dural Align LLC, New York, USA) was employed to create a montage of AO images, with the objective of identifying the foveal center. This was achieved by superimposing the AO images with SLO and OCT images from Spectralis.\u003c/p\u003e \u003cp\u003eFour ROIs were strategically positioned at 2\u0026deg; and 4\u0026deg; in the temporal and nasal directions, respectively. The ROI was obtained from superior and inferior points, and the CDs at these points were subsequently averaged (indicated by red squares in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For an AL of 24 mm, image size of 62 \u0026times; 62 \u0026micro;m was determined[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We used the follow-up mode of AOdetect to analyze and measure the CD from identical ROIs. The ROIs were selected using the maximum density method, which has been described previously[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Measurements were corrected based on each participant\u0026rsquo;s AL. The software calculated local cell density (cells/mm\u0026sup2;) and regularity (%) through the Voronoi analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Given that AO imaging may misidentify hyperreflective spots as cone photoreceptors, automated cone detection results were manually verified and corrected by two retinal specialists.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOCT analysis\u003c/h2\u003e \u003cp\u003eOCT imaging was performed using the Spectralis device (Heidelberg Engineering, Heidelberg, Germany). EZ length was measured in both eyes using the \"caliper\" function of the internal software (Heidelberg Eye Explorer version 6.12.3.0), and the same OCT images used for AO analysis were employed for this measurement. The analysis involved horizontal OCT scans aligned with the ROIs defined by AO images. Three ophthalmologists performed the measurements, and the average of their readings constituted the final measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHumphrey Field Analyzer analysis\u003c/h2\u003e \u003cp\u003eAll patients underwent automated static perimetry using the HF10-2 SITA standard program. Reliability criteria included a fixation loss score of \u0026le;\u0026thinsp;20%, with false-positive or false-negative rate set at \u0026le;\u0026thinsp;33%. Data were excluded from the study if repeat tests did not yield reliable results. Considering that dB values are logarithmically scaled, averaging dB sensitivity across points was deemed inappropriate[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, dB sensitivity values were first converted to 1/Lambert, averaged, and then transformed back into the dB scale as \"antilogged first average (dB)\" for analysis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Each TD value was first antilogged and then averaged. To return to the dB scale, the values were log-transformed again, yielding the \u0026ldquo;antilogged first average (ALFA)\u0026rdquo; (dB) values. ALFA was analyzed for both the overall average (68 points were used for TD analysis [TD\u003csub\u003e68\u003c/sub\u003e]) and the central four points (C4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eComparisons between visits were conducted using paired t-tests with Bonferroni correction for multiple comparisons. To evaluate longitudinal changes in CD, LMMs were applied separately for each ROI to estimate the annual rate of decline and to assess whether VISIT had a statistically significant effect on CD. P-values for the fixed effects in the LMMs were calculated using Satterthwaite\u0026rsquo;s approximation. Associations between CD and other parameters (EZ, MD, VA, C4, and TD\u003csub\u003e68\u003c/sub\u003e) were assessed using Pearson\u0026rsquo;s correlation coefficients. All measurements were independently evaluated by two ophthalmologists. For the correlation analyses, all available data points were included, even in instances where all five visits were not completed, provided the two variables of interest were measured at the same visit. Statistical analyses were performed using Microsoft Excel version 16.16.27 (Microsoft, Redmond, USA) and RStudio version 2024.04.2\u0026thinsp;+\u0026thinsp;764.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics declaration\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Research Review Committee of Kobe City Kobe Eye Center Hospital (Permit No.: ek241201) and by the Institutional Review Board at the Research Committee of Santen Pharmaceutical Co., Ltd. The study was registered at the University Hospital Medical Information Network Clinical Trials Registry (clinical trial identifier: UMIN000057025).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.M. received a financial support from Santen. Y.T. and M.T. are employees of Santen. H.M., M.Y., E.G., S.Y., S.K., Y.H., Y.K., and K.G. declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003eDeclaration\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Santen Pharmaceutical Co., Ltd. The funder had no role in study design, data collection, data analysis, or manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore collecting the samples from the patients, written informed consent was obtained in accordance with the guidelines of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Santen. We thank the faculty and staff of Kobe City Eye Hospital and Kawasaki Medical School for their comments and discussion, the members of Imagine Eyes and Santen Pharmaceutical Co., Ltd. for their support, and Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.G. and A.M. designed and conducted the study. K.G., M.Y., E.G., S.K., S.Y., Y.T., and H.M. performed the data acquisition and analysis, and H.M. drafted the manuscript. M.T., Y.H., and Y.K. provided critical input during manuscript preparation. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetic variant data generated in this study have been deposited in ClinVar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(https://www.ncbi.nlm.nih.gov/clinvar/) under the accession numbers\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSCV007122111 to SCV007122125.\u003c/p\u003e\n\u003cp\u003eAll other data supporting the findings of this study are included within the article\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eand its Supplementary Information files. \u0026nbsp;The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Supplementary figures and tables associated with the main findings are provided in the Supplementary Information file.\u003cstrong\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eConsugar, M. B. et al. Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing. \u003cem\u003eGenet. Med.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 253\u0026ndash;261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/gim.2014.172\u003c/span\u003e\u003cspan address=\"10.1038/gim.2014.172\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePieras, J. I. et al. Copy-number variations in EYS: a significant event in the appearance of arRP. \u003cem\u003eInvest. Ophthalmol. Vis. 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Rep.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 16383. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-017-16640-7\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-16640-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"adaptive optics ophthalmoscopy, photoreceptor, retinitis pigmentosa, inherited retinal dystrophy","lastPublishedDoi":"10.21203/rs.3.rs-8198915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8198915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was conducted to evaluate the longitudinal morphological changes in cone density (CD) in patients with the eyes shut homolog (\u003cem\u003eEYS\u003c/em\u003e)-associated retinitis pigmentosa (RP) using adaptive optics (AO) fundus imaging and to assess its potential as a biomarker for disease progression. As a sub-analysis of the KEYS study, this prospective observational study was conducted at the Kobe Eye Center, involving 50 patients with \u003cem\u003eEYS\u003c/em\u003e-RP; 27 eyes from 27 patients who were eligible for adaptive optics fundus imaging were included in this analysis. Ellipsoid zone (EZ) length was assessed using optical coherence tomography, and mean deviation (MD) values were obtained from static visual field. CD showed a significant reduction in all regions of interest as early as 6 months from baseline. In contrast, a significant decrease in EZ length was observed only at 24 months, while MD values did not exhibit significant changes throughout the observation period. AO fundus imaging demonstrated high sensitivity in detecting early structural changes in \u003cem\u003eEYS\u003c/em\u003e-RP. These findings contribute to a deeper understanding of the natural history of the disease and suggest that CD measurement could serve as a valuable biomarker for monitoring disease progression and evaluating treatment efficacy.\u003c/p\u003e","manuscriptTitle":"Two-year prospective natural history study of EYS-associated retinitis pigmentosa using adaptive optics: The KEYS Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 09:23:33","doi":"10.21203/rs.3.rs-8198915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-21T15:33:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T21:32:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328122519435023683879834442239589260920","date":"2026-03-10T20:14:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-08T10:44:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267349393340351258076467911311675237024","date":"2026-03-02T16:30:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T10:38:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-25T04:43:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T10:30:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-21T08:46:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-21T08:40:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ddf39610-e5f7-47c4-a551-e58dc4228409","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63545325,"name":"Health sciences/Biomarkers"},{"id":63545326,"name":"Health sciences/Diseases"},{"id":63545327,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-28T23:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 09:23:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8198915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8198915","identity":"rs-8198915","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}