Clinical and preclinical therapeutic outcome metrics for USH2A-related disease.

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease.

Investigating Biomarkers for USH2A Retinopathy Using Multimodal Retinal Imaging.

Pathogenic mutations in USH2A are a leading cause of visual loss secondary to non-syndromic or Usher syndrome-associated retinitis pigmentosa (RP). With an increasing number of RP-targeted clinical trials in progress, we sought to evaluate the photoreceptor topography underlying patterns of loss observed on clinical retinal imaging to guide surrogate endpoint selection in USH2A retinopathy. In this prospective cross-sectional study, twenty-five patients with molecularly confirmed USH2A-RP underwent fundus autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) retinal imaging. Analysis comprised measurement of FAF horizontal inner (IR) and outer (OR) hyperautofluorescent ring diameter; SD-OCT ellipsoid zone (EZ) and external limiting membrane (ELM) width, normalised EZ reflectance; AOSLO foveal cone density and intact macular photoreceptor mosaic (IMPM) diameter. Thirty-two eyes from 16 patients (mean age ± SD, 36.0 ± 14.2 years) with USH2A-associated Usher syndrome type 2 (n = 14) or non-syndromic RP (n = 2) met the inclusion criteria. Spatial alignment was observed between IR-EZ and OR-ELM diameters/widths (p < 0.001). The IMPM border occurred just lateral to EZ loss (p < 0.001), although sparser intact photoreceptor inner segments were detected until ELM disruption. EZ width and IR diameter displayed a biphasic relationship with cone density whereby slow cone loss occurred until retinal degeneration reached ~1350 µm from the fovea, beyond which greater reduction in cone density followed. Normalised EZ reflectance and cone density were significantly associated (p < 0.001). As the strongest correlate of cone density (p < 0.001) and best-corrected visual acuity (p < 0.001), EZ width is the most sensitive biomarker of structural and functional decline in USH2A retinopathy, rendering it a promising trial endpoint.

Characteristics of Retinitis Pigmentosa Associated with ADGRV1 and Comparison with USH2A in Patients from a Multicentric Usher Syndrome Study Treatrush.

In contrast to USH2A, variants in ADGRV1 are a minor cause of Usher syndrome type 2, and the associated phenotype is less known. The purpose of the study was to characterize the retinal phenotype of 18 ADGRV1 patients (9 male, 9 female; median age 52 years) and compare it with that of 204 USH2A patients (111 male, 93 female; median age 43 years) in terms of nyctalopia onset, best corrected visual acuity (BCVA), fundus autofluorescence (FAF), and optical coherence tomography (OCT) features. There was no statistical difference in the median age at onset (30 and 18 years; Mann-Whitney U test, p = 0.13); the mean age when 50% of the patients reached legal blindness (≥1.0 log MAR) based on visual acuity (64 years for both groups; log-rank, p = 0.3); the risk of developing advanced retinal degeneration (patch or atrophy) with age (multiple logistic regression, p = 0.8); or the frequency of cystoid macular edema (31% vs. 26%, Fisher's exact test, p = 0.4). ADGRV1 and USH2A retinopathy were indistinguishable in all major functional and structural characteristics, suggesting that the loss of function of the corresponding proteins produces similar effects in the retina. The results are important for counseling ADGRV1 patients, who represent the minor patient subgroup.

Biomarkers in Usher syndrome: ultra-widefield fundus autofluorescence and optical coherence tomography findings and their correlation with visual acuity and electrophysiology findings.

PURPOSE: To investigate the functional and structural biomarkers and their correlation with Usher syndrome (USH). METHODS: Medical records, imaging and electrophysiology test results of USH patients attending the Save Sight Institute between 2012 and 2017 were reviewed. Best corrected visual acuity (BCVA), ultra-widefield autofluorescence (UW-FAF), spectral-domain optical coherence tomography (SD-OCT), full-field electroretinogram and pattern electroretinogram (pERG) were performed. SD-OCT scans assessed central macular thickness (CMT), greatest linear diameter of preserved outer retinal layers-macular island (MI) and presence of cystoid macular edema (CME). UW-FAF images were qualitatively graded to identify hypo/hyperfluorescence patterns in the peripheral fundus. RESULTS: Thirty-six eyes from 18 subjects were included. Mean BCVA was 0.22 ± 0.3 LogMAR. MI extent was significantly associated with better vision (ß = - 0.175 per 1000 µm; R2 = 0.487; P = 0.002; Fig. 4). A higher pERG P50 was associated with a larger macular island (ß = 782 per µV; R2 = 0.238; P = 0.025), while a higher pERG N95 was associated with a smaller macular island (ß = - 499 per µV; R2 = 0.219; P = 0.030). Mean CMT was 271 ± 35 µm and was significantly associated with better vision (ß = - 0.083 per 10 µm; R2 = 0.612; P < 0.001). CME was diagnosed in 47.2% (n = 17) eyes. There was no significant difference in mean BCVA for those with CME (0.19 ± 0.2 LogMAR) and without CME (0.40 ± 0.5; R2 = 0.081; P = 0.17). All patients had abnormal UW-FAF. Four main patterns of change were identified (granular 55%, annular 11%, bone spicule 17% and patchy 17%). Patients with the patchy pattern demonstrated worse BCVA in comparison with those with granular (P < 0.0001) and bone spicule (P = 0.0179) patterns. CONCLUSIONS: Structural changes identified on OCT and UW-FAF correlated with BCVA and pERG in this cohort representing different stages of the disease. These parameters could represent reliable biomarkers in therapeutic clinical trials on USH.

Multimodal photoacoustic microscopy, optical coherence tomography, and fluorescence imaging of USH2A knockout rabbits.

Usher syndrome type 2A (USH2A) is a genetic disorder characterized by retinal degeneration and hearing loss. To better understand the pathogenesis and progression of this syndrome, animal models such as USH2A knockout (USH2AKO) rabbits have been developed. In this study, we employed multimodal imaging techniques, including photoacoustic microscopy (PAM), optical coherence tomography (OCT), fundus autofluorescence (FAF), fluorescein angiography (FA), and indocyanine green angiography (ICGA) imaging to evaluate the retinal changes in the USH2AKO rabbit model. Twelve New Zealand White rabbits including USH2AKO and wild type (WT) were used for the experiments. Multimodal imaging was implemented at different time points over a period of 12 months to visualize the progression of retinal changes in USH2AKO rabbits. The results demonstrate that ellipsoid zone (EZ) disruption and degeneration, key features of Usher syndrome, began at the age of 4 months old and persisted up to 12 months. The EZ degeneration areas were clearly observed on the FAF and OCT images. The FAF images revealed retinal pigment epithelium (RPE) degeneration, confirming the presence of the disease phenotype in the USH2AKO rabbits. In addition, PAM images provided high-resolution and high image contrast of the optic nerve and the retinal microvasculature, including retinal vessels, choroidal vessels, and capillaries in three-dimensions. The quantification of EZ fluorescent intensity using FAF and EZ thickness using OCT provided comprehensive quantitative data on the progression of degenerative changes over time. This multimodal imaging approach allowed for a comprehensive and non-invasive assessment of retinal structure, microvasculature, and degenerative changes in the USH2AKO rabbit model. The combination of PAM, OCT, and fluorescent imaging facilitated longitudinal monitoring of disease progression and provided valuable insights into the pathophysiology of USH2A syndrome. These findings contribute to the understanding of USH2A syndrome and may have implications for the development of diagnostic and therapeutic strategies for affected individuals. The multimodal imaging techniques employed in this study offer a promising platform for preclinical evaluation of potential treatments and may pave the way for future clinical applications in patients with Usher syndrome.

Phenotypic and Genetic Characteristics in a Cohort of Patients with Usher Genes.

Background: This study aimed to compare phenotype−genotype correlation in patients with Usher syndrome (USH) to those with autosomal recessive retinitis pigmentosa (NS-ARRP) caused by genes associated with Usher syndrome. Methods: Case notes of patients with USH or NS-ARRP and a molecularly confirmed diagnosis in genes associated with Usher syndrome were reviewed. Phenotypic information, including the age of ocular symptoms, hearing impairment, visual acuity, Goldmann visual fields, fundus autofluorescence (FAF) imaging and spectral domain optical coherence tomography (OCT) imaging, was reviewed. The patients were divided into three genotype groups based on variant severity for genotype-phenotype correlations. Results: 39 patients with Usher syndrome and 33 patients with NS-ARRP and a molecular diagnosis in an Usher syndrome-related gene were identified. In the 39 patients diagnosed with Usher syndrome, a molecular diagnosis was confirmed as follows: USH2A (28), MYO7A (4), CDH23 (2), USH1C (2), GPR98/VLGR1 (2) and PCDH15 (1). All 33 patients with NS-ARRP had variants in USH2A. Further analysis was performed on the patients with USH2A variants. USH2A patients with syndromic features had an earlier mean age of symptom onset (17.9 vs. 31.7 years, p < 0.001), had more advanced changes on FAF imaging (p = 0.040) and were more likely to have cystoid macular oedema (p = 0.021) when compared to USH2A patients presenting with non-syndromic NS-ARRP. Self-reported late-onset hearing loss was identified in 33.3% of patients with NS-ARRP. Having a syndromic phenotype was associated with more severe USH2A variants (p < 0.001). Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort. Conclusions: Patients with Usher syndrome, whatever the associated gene in this cohort, tended to have an earlier onset of retinal disease (other than GPR98/VLGR1) when compared to patients presenting with NS-ARRP. Analysis of genetic variants in USH2A, the commonest gene in our cohort, showed that patients with a more severe genotype were more likely to be diagnosed with USH compared to NS-ARRP. USH2A patients with syndromic features have an earlier onset of symptoms and more severe features on FAF and OCT imaging. However, a third of patients diagnosed with NS-ARRP developed later onset hearing loss. Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort, thus expanding the genetic spectrum of known pathogenic variants. An accurate molecular diagnosis is important for diagnosis and prognosis and has become particularly relevant with the advent of potential therapies for Usher-related gene

Decreased activity of piriform cortex and orbitofrontal hyperactivation in Usher Syndrome, a human disorder of ciliary dysfunction.

Usher syndrome (USH) is a condition characterized by ciliary dysfunction leading to retinal degeneration and hearing/vestibular loss. Putative olfactory deficits in humans have been documented at the psychophysical level and remain to be proven at the neurophysiological level. Thus, we aimed to study USH olfactory impairment using functional magnetic resonance imaging. We analyzed differences in whole-brain responses between 27 USH patients and 26 healthy participants during an olfactory detection task with a bimodal odorant (n-butanol). The main research question was whether between-group differences could be identified using a conservative whole-brain approach and in a ROI-based approach in key olfactory brain regions. Results indicated higher olfactory thresholds in USH patients, thereby confirming the hypothesis of reduced olfactory acuity. Importantly, we found decreased BOLD activity for USH patients in response to odorant stimulation in the right piriform cortex, while right orbitofrontal cortex showed increased activity. We also found decreased activity in other higher-level regions in a whole brain approach. We suggest that the hyper activation in the orbitofrontal cortex possibly occurs as a compensatory mechanism after the under-recruitment of the piriform cortex. This study suggests that olfactory deficits in USH can be objectively assessed using functional neuroimaging which reveals differential patterns of activity both in low- and high-level regions of the olfactory network.

Multimodal imaging and genetic findings in a case of ARSG-related atypical Usher syndrome.

Background: Atypical Usher syndrome has recently been associated with arylsulfatase G (ARSG) variants. In these cases, characteristic findings include progressive sensorineural hearing loss (SNHL) without vestibular involvement and ring-shaped late-onset retinitis pigmentosa (RP).Materials and Methods: One patient with atypical Usher syndrome and a novel homozygous ARSG variant was included in this study. The patient underwent a comprehensive ophthalmic examination, including multimodal imaging and genetic testing.Results: A 60-year-old male of Persian decent presented to our clinic with a history of 20 years of progressive SNHL, and 10 years of progressive peripheral vision loss and pigmentary retinopathy. Consistent with previous reports of ARSG-related atypical Usher syndrome, fundus examination revealed ring-shaped retinal hyperpigmentation and fundus autofluorescence (FAF) demonstrated a six-zone pattern of autofluorescence. Optical coherence tomography (OCT) showed extensive cystoid spaces concentrated in the ganglion cell layer. Widefield OCT angiography at the level of the choriocapillaris showed signs of atrophy that corresponded to the FAF hypofluorescent zone. The patient was homozygous for a novel ARSG variant c. 1270 C > T, p. Arg424Cys.Conclusion: We report a novel ARSG variant in a case of atypical Usher syndrome and describe multimodal imaging findings that further characterize the effect of ARSG in the pathogenesis of atypical Usher syndrome.

Expanding the clinical phenotype in patients with disease causing variants associated with atypical Usher syndrome.

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG, and ABHD12.Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG, or ABHD12.CEP78-related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12-related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe.This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH.

Characterisation of microvascular abnormalities using OCT angiography in patients with biallelic variants in USH2A and MYO7A.

AIMS: Using optical coherence tomography angiography (OCTA) to characterise microvascular changes in the retinal plexuses and choriocapillaris (CC) of patients with MYO7A and USH2A mutations and correlate with genotype, retinal structure and function. METHODS: Twenty-seven patients with molecularly confirmed USH2A (n=21) and MYO7A (n=6) mutations underwent macular 6×6 mm OCTA using the AngioVue. Heidelberg spectral-domain OCT scans and MAIA microperimetry were also performed, the preserved ellipsoid zone (EZ) band width and mean macular sensitivity (MS) were recorded. OCTA of the inner retina, superficial capillary plexus (SCP), deep capillary plexus (DCP) and CC were analysed. Vessel density (VD) was calculated from the en face OCT angiograms of retinal circulation. RESULTS: Forty-eight eyes with either USH2A (n=37, mean age: 34.4±12.2 years) or MYO7A (n=11, mean age: 37.1±12.4 years), and 35 eyes from 18 age-matched healthy participants were included. VD was significantly decreased in the retinal circulation of patients with USH2A and MYO7A mutations compared with controls (p<0.001). Changes were observed in both the SCP and DCP, but no differences in retinal perfusion were detected between USH2A and MYO7A groups. No vascular defects were detected in CC of the USH2A group, but peripheral defects were detected in older MYO7A patients from the fourth decade of life. VD in the DCP showed strong association with MS and EZ width (Spearman's rho =0.64 and 0.59, respectively, p<0.001). CONCLUSION: OCTA was able to detect similar retinal microvascular changes in patients with USH2A and MYO7A mutations. The CC was generally affected in MYO7A mutations. OCT angiography may further enhance our understanding of inherited eye diseases and their phenotype-genotype associations.

Double Hyperautofluorescent Rings in Patients with USH2A-Retinopathy.

USH2A mutation is the most common cause of retinitis pigmentosa, with or without hearing impairment. Patients most commonly exhibit hyperautofluorescent ring on fundus autofluorescence imaging (FAF) and rod-cone dystrophy on electrophysiology. A detailed study of three USH2A patients with a rare pattern of double hyperautofluorescent rings was performed. Twenty-four patients with typical single hyperautofluorescent rings were used for comparison of the ages of onset, visual fields, optical coherence tomography, electrophysiology, and audiograms. Double rings delineated the area of pericentral retinal degeneration in all cases. Two patients exhibited rod-cone dystrophy, whereas the third had a cone-rod dystrophy type of dysfunction on electrophysiology. There was minimal progression on follow-up in all three. Patients with double rings had significantly better visual acuity, cone function, and auditory performance than the single ring group. Double rings were associated with combinations of null and missense mutations, none of the latter found in the single ring patients. According to these findings, the double hyperautofluorescent rings indicate a mild subtype of USH2A disease, characterized by pericentral retinal degeneration, mild to moderate hearing loss, and either a rod-cone or cone-rod pattern on electrophysiology, the latter expanding the known clinical spectrum of USH2A-retinopathy.

Evidence for impaired olfactory function and structural brain integrity in a disorder of ciliary function, Usher syndrome.

Diseases involving cilia dysfunction, such as Usher Syndrome (USH), often involve visual and auditory loss. Psychophysical evidence has suggested that this may also hold true for the peripheral olfactory domain. Here we aimed to go a step further by attempting to establish relations between the integrity of cortical structures and olfactory function in this condition. We investigated olfactory function for USH types 1 (USH1) and 2 (USH2). Bilateral olfactory bulb (OB) volume and olfactory sulcus (OS) depth were also analysed. Thirty-three controls with no previous olfactory deficits were age, sex and handedness-matched to 32 USH patients (11 USH1, 21 USH2). A butanol detection threshold test was performed to measure olfactory function. For OB volume and OS depth, morphometric measurements were performed using magnetic resonance imaging (MRI) based on detailed segmentation by three independent operators. Averaged values across these were used for the statistical analyses. Total intracranial volume was estimated using Freesurfer to account for head size variability. Olfactory threshold was significantly lower in controls when compared to USH, USH1, and USH2. OS depth was found to be shallower in both hemispheres in USH patients when compared with the control group. OB volume was not significantly different between control and USH groups, or respective subgroups. Nevertheless, butanol threshold was negatively correlated with the left OB volume for the USH type 1 subgroup. The main effect of OS depth reduction was found to be mainly due to the comparison between USH2 and controls. Our results provide evidence for morphometric changes and olfactory dysfunction in patients with USH. This correlated with a reduction in left OB volume in the USH1 subgroup, the most severe USH phenotype. The main effect of reduced OS depth was found to stem mainly from USH2 raising questions regarding a possible complex interaction between sensory olfactory loss and central cortical changes in this disease.

Histopathological and neuroradiological features of Usher syndrome type II.

OBJECTIVE: To study the histopathological and neuroradiological features of Usher syndrome (USH), with particular focus on USH type II, an inherited disorder characterized by moderate to severe congenital hearing impairment and retinitis pigmentosa with onset in the late teens. METHODOLOGY: A report of four cases and literature review. RESULTS AND CONCLUSION: Rare examples of histopathological and neuroradiological findings from four USH type II cases are presented. More studies like these are encouraged so that correlation studies between the morphological and clinical findings can be performed on the path to elucidate the pathogenesis of this heterogeneous disorder.

Prevalence of macular abnormalities assessed by optical coherence tomography in patients with Usher syndrome.

BACKGROUND: To investigate the prevalence of macular abnormalities in patients affected by Usher syndrome (USH), by comparing the clinical findings between two types (i.e., USH1 and USH2). MATERIAL AND METHODS: A retrospective study was performed by reviewing optical coherence tomography (OCT) in 134 USH patients to determine the presence of macular abnormalities, including cystoid macular edema (CME), epiretinal membrane (ERM), vitreo-macular traction syndrome (VMT), and macular hole (MH). RESULTS: Macular abnormalities were observed in 126/268 (47.0%) examined eyes. The most frequent abnormality was ERM observed in 51 eyes (19%), followed by CME observed in 42 eyes (15.7%). Moreover, CME was significantly (p < 0.05) associated with younger age (CME: 30.1 ± 11.1 years; without CME: 36.9 ± 14.9 years), whereas VMT and full thickness MH were associated with older age (p < 0.05). Moreover, a significantly (p < 0.05) decreased best-corrected visual acuity was associated with MH compared to eyes without MH. Finally, CME was more frequent in USH1 compared to USH2. CONCLUSION: Our study, for the first time in the literature, showed the distribution of all macular abnormalities assessed by SD-OCT in a large USH cohort, comparing USH1 and USH2 patients. We observed that ocular abnormalities are highly prevalent in USH patients compared to general population, with ERM and CME being the most common alterations. Based on these findings, OCT screening in USH patients is recommended for early detection of macular changes and early treatment.