Non-syndromic OTX2-associated pattern dystrophy: a 10-year multimodal imaging study.

PURPOSE: To report novel multimodal imaging features and long-term follow-up of Orthodenticle Homeobox 2 (OTX2)-associated pattern Gdystrophy. METHODS: A 14-year-old boy referred with glaucoma suspect and macular pigmentation underwent fundus autofluorescence imaging, optical coherence tomography, fluorescein and indocyanine green angiography, visual field test, microperimetry and electrophysiology over a ten-year period. Next-generation sequencing panel identified a de novo heterozygous likely pathogenic OTX2 variant, c.259G>A, [p.(Glu87Lys)]. RESULTS: Visual acuity was 20/40 OD and 20/30 OS. Examination showed bilateral enlarged optic nerve heads and increased disc cupping, multiple cilioretinal arteries, a pigmentary maculopathy with stellate-shaped region of hypoautofluorescence, shallow serous macular detachment, subretinal deposits and temporal avascular retina. Angiography showed no source of leakage and absence of retinal neovascularisation despite extensive peripheral non perfusion. Electrophysiological assessments demonstrated mild progressive rod and cone pathway abnormalities, reduced light-adapted b:a ratio, and reduced Arden ratio on electro-oculogram. Ten-year follow-up confirmed a stable disease course despite persistent submacular fluid. There was no associated pituitary structural abnormality or dysfunction. CONCLUSIONS: This case study contributes to further understanding of OTX2-associated pattern dystrophy, highlighting its stability over 10 years. Further investigation into inter-individual and intrafamilial variability is warranted.

Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

SIBLING CONCORDANCE IN SYMPTOM ONSET AND ATROPHY GROWTH RATES IN STARGARDT DISEASE USING ULTRA-WIDEFIELD FUNDUS AUTOFLUORESCENCE.

PURPOSE: To investigate concordance in symptom onset, area of dark autofluorescence (DAF), and growth rate (GR) between Stargardt disease siblings at an age-matched time point. METHODS: In this retrospective longitudinal study of sibling pairs with identical biallelic ABCA4 variants, age at symptom onset, best-corrected visual acuity, atrophy area, and effective radius of DAF on ultra-widefield fundus autofluorescence were recorded. Absolute intersibling differences for both eyes were compared with absolute interocular differences using the Mann-Whitney test. RESULTS: Overall 39 patients from 19 families were recruited. In 16 families, age-matched best-corrected visual acuity and DAF were compared between siblings. In 8 families, DAF GR was compared. The median (range) absolute difference in age at symptom onset between siblings was 3 (0-35) years. Absolute intersibling differences in age-matched best-corrected visual acuity were greater than interocular differences ( P = 0.01). Similarly, absolute intersibling differences in DAF area and radius were greater than interocular differences ( P = 0.04 for area and P = 0.001 for radius). Differences between absolute interocular and intersibling GR were not statistically significant ( P = 0.44 for area GR and P = 0.61 for radius GR). CONCLUSION: There was significant discordance in age-matched best-corrected visual acuity and DAF beyond the expected limits of interocular asymmetry. Lack of significant intersibling differences in GR warrants further investigation.

A novel phenotype in a family with autosomal dominant retinal dystrophy due to c.1430A > G in retinoid isomerohydrolase (RPE65) and c.37C > T in bestrophin 1 (BEST1).

PURPOSE: The c.1430A > G (Asp477Gly) variant in RPE65 has been reported in Irish and Scottish families with either an autosomal dominant retinal dystrophy (adRD) that resembles choroideremia, a vitelliform macular dystrophy or an isolated macular atrophy. We report novel features on multimodal imaging and the natural history of a family harbouring this variant in combination with the BEST1 c.37C > T (Arg13Cys) variant. METHODS: Members of a family with an adRD were examined clinically to ascertain phenotype and underwent genetic testing. Multimodal imaging included widefield colour fundus photography, quantitative autofluorescence (qAF) and spectral domain optical coherence tomography. Electrophysiology and microperimetry were also performed. RESULTS: Vision loss was attributed to foveal atrophy in the proband and choroidal neovascularisation and a vitello-eruptive lesion in one affected son. Peripheral retinal white dots corresponding to subretinal deposits were seen in three patients. The median qAF8 values in the proband (I:1) were low (40 and 101 in OD and OS) at age 79. Similarly, the qAF8 values for the middle son (II:2) were also low (100 and 87 in ODS and OS) at age 60. Electrophysiology showed disproportionate reduction in Arden ratio prior to the gradual loss of full-field responses. Microperimetry demonstrated an enlarging scotoma in the proband. CONCLUSIONS: The coexistence of the pathogenic BEST1 c.37C > T variant may modify clinical features observed in RPE65 adRD. This study expands our understanding of RPE65 adRD as a retinoid cycle disorder supported by the reduced qAF, fine white retinal dots and corresponding subretinal deposits on OCT in affected members.

CLASSIFYING ABCA4 MUTATION SEVERITY USING AGE-DEPENDENT ULTRA-WIDEFIELD FUNDUS AUTOFLUORESCENCE-DERIVED TOTAL LESION SIZE.

PURPOSE: To establish a mutation-specific age-dependent ultra-widefield fundus autofluorescence (UWF-FAF) trajectory in a large Stargardt disease (STGD1) cohort using total lesion size (TLS) and to develop a clinical method for variant classification. METHODS: A retrospective study of patients with biallelic ABCA4 mutations that were evaluated with UWF-FAF. Boundaries of TLS, defined by stippled hyper/hypoautofluorescence, were outlined manually. Pathogenicity was assessed according to ACMG/AMP criteria, and mutation severities were classified based on the current literature. Age-dependent trajectories in TLS were examined in patients with nullizygous, mild, and intermediate mutations. Mutations of uncertain severities were classified using a clinical criterion based on age of symptom onset and TLS. RESULTS: Eighty-one patients with STGD1 (mean age = 42 ± 20 years and mean visual acuity = 20/200) were recruited from 65 unrelated families. Patients with biallelic null/severe variants (n = 6) demonstrated an increase in TLS during their second decade reaching a mean ± SD of 796 ± 29 mm2 by age 40. Those harboring mild mutations c.5882G>A or c.5603A>T had lesions confined to the posterior pole with a mean ± SD TLS of 30 ± 39 mm2. Intermediate mutations c.6079C>T or c.[2588G>C;5603A>T] in trans with a null/severe mutation had a mean ± SD TLS of 397 ± 29 mm2. Thirty-two mutations were predicted to cause severe (n = 22), intermediate (n = 6), and mild (n = 5) impairment of ABCA4 function based on age of symptom onset and TLS. CONCLUSION: Age-dependent TLS showed unique ABCA4 mutation-specific trajectories. Our novel clinical criterion using age of symptom onset and TLS to segregate ABCA4 mutations into three severity groups requires further molecular studies to confirm its validity.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Clinical and molecular characterization of non-syndromic retinal dystrophy due to c.175G>A mutation in ceroid lipofuscinosis neuronal 3 (CLN3).

PURPOSE: Mutation of the CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, has recently been associated with late-onset, non-syndromic retinal dystrophy. Herein we describe the multimodal imaging, immunological and systemic features of an adult with compound heterozygous CLN3 mutations. METHODS: A 50-year-old female with non-syndromic retinal dystrophy from the age of 36 years underwent multimodal retinal imaging, electroretinography, neuroimaging, immunological studies and genetic testing. CLN3 transcripts were amplified from patient leukocytes by reverse transcriptase polymerase chain reaction and characterized by Sanger sequencing. RESULTS: Visual acuity declined to 6/12 and 6/76 due to asymmetrical central scotoma. ERG responses became electronegative and patient's serum contained anti-retinal antibodies. Final visual acuity stabilized at 6/60 bilaterally 3 years after peri-ocular steroid and rituximab infusion. Genetic testing revealed compound heterozygous CLN3 mutations: the 1.02 kb deletion and a novel missense mutation (c.175G>A). In silico, analyses predicted the c.175G>A mutation disrupted an exonic splice enhancer site in exon 3. In patient leukocytes, CLN3 expression was reduced and novel CLN3 transcripts lacking exon 3 were detected. CONCLUSIONS: Our case study shows that (1) non-syndromic CLN3 disease leads to rod and delayed primary cone degeneration resulting in constricting peripheral field and enlarging central scotoma and, (2) the c.175G>A CLN3 mutation, altered splicing of the CLN3 gene. Overall, we provide comprehensive clinical characterization of a patient with non-syndromic CLN3 disease.

Development and Psychometric Assessment of Novel Item Banks for Hereditary Retinal Diseases.

SIGNIFICANCE: This study develops psychometrically valid item banks across 10 areas of quality of life (QoL) specific to people with hereditary retinal diseases, which will enable clinicians and researchers to explore the impact of hereditary retinal diseases across all aspects of QoL. PURPOSE: The purpose of this study was to assess the psychometric properties of hereditary retinal disease QoL item banks using Rasch analysis and demonstrate the effectiveness of a computerized adaptive testing (CAT) system in obtaining precise measurement of QoL using only a few items. METHODS: The hereditary retinal disease item banks were answered by 233 participants (median age, 58 years; range, 18 to 94 years; female participants, 59%). The hereditary retinal disease item banks cover 10 QoL domains: activity limitation, mobility, emotional, social, convenience, economic, health concerns, visual symptoms, ocular comfort symptoms, and general symptoms. Rasch analysis assessed the psychometric properties of the 10 item banks and provided item calibrations for the development of CAT. Computerized adaptive testing simulations were performed to calculate the average number of items required to gain precise measurement of each QoL domain. RESULTS: The convenience, economic, visual symptoms, and the social domains formed unidimensional scales. However, the activity limitation and health concerns domains demonstrated multidimensionality and required major modifications to resolve this, which resulted in four new QoL domains, namely, reading, driving, lighting, and concerns about the disease progression. In total, 10 item banks underwent CAT simulation testing, which indicated that 8 to 12 items were required to gain precise measurement of each QoL domain. CONCLUSIONS: We have developed 10 psychometrically valid item banks to measure the QoL domains relevant to people with hereditary retinal diseases. On average, only 5 and 10 items were required to gain measurement at moderate and high precision, respectively.

Analysis of the ABCA4 c.[2588G>C;5603A>T] Allele in the Australian Population.

Inherited retinal diseases (IRDs) are genetically and phenotypically diverse, and they cause significant morbidity worldwide. Importantly, IRDs may be amenable to precision medicine strategies, and thus the molecular characterisation of causative variants is becoming increasingly important with the promise of personalised therapies on the horizon. ABCA4, involved in the translocation of visual cycle derivatives, is a well-established, frequent cause of IRDs worldwide, with pathogenic variants implicated in phenotypically diverse diseases. Identification of causative ABCA4 variants in some individuals, however, has been enigmatic, and resolution of this issue is currently a hotbed of research. Recent evidence has indicated that hypomorphic alleles, which cause disease under certain conditions, may account for some of the missing causal variants. It has been postulated that the ABCA4 c.5603A>T (p.Asn1868Ile) variant, previously considered benign, be reclassified as hypomorphic when in cis configuration with c.2588G>C (p.Gly863Ala/Gly863del), a variant previously considered to be pathogenic in its own right. We are exploring this relationship within an Australian cohort to test this theory.

Maternal uniparental isodisomy of chromosome 6 unmasks a novel variant in TULP1 in a patient with early onset retinal dystrophy.

Purpose: Inherited retinal dystrophies are a clinically and genetically heterogeneous group of disorders. Molecular diagnosis has proven utility for affected individuals. In this study, we report an individual enrolled in the Australian Inherited Retinal Disease Registry and DNA Bank diagnosed with clinical features overlapping between Leber congenital amaurosis and retinitis pigmentosa. Methods: DNA from the proband was sequenced using a gene panel for inherited retinal disorders, and a single nucleotide polymorphism (SNP) array was conducted to detect the presence of deletions and uniparental disomy. Results: We identified a novel homozygous variant (c.524dupC, p.(Pro176ThrfsTer7)) in TULP1 resulting from maternal uniparental isodisomy of chromosome 6. The patient had clinical features consistent with biallelic pathogenic variants in TULP1, including congenital nystagmus, night blindness, non-recordable electroretinogram, mild myopia, and mild peripheral pigmentary changes in the fundus. Conclusions: This is the first report of uniparental disomy 6 and a homozygous variant in TULP1 associated with a rod-cone dystrophy. Molecular diagnosis of inherited retinal dystrophies is essential to inform the mode of transmission and clinical management, and to identify potential candidates for future gene-specific therapies.

Developing an item bank to measure the coping strategies of people with hereditary retinal diseases.

PURPOSE: Our understanding of the coping strategies used by people with visual impairment to manage stress related to visual loss is limited. This study aims to develop a sophisticated coping instrument in the form of an item bank implemented via Computerised adaptive testing (CAT) for hereditary retinal diseases. METHODS: Items on coping were extracted from qualitative interviews with patients which were supplemented by items from a literature review. A systematic multi-stage process of item refinement was carried out followed by expert panel discussion and cognitive interviews. The final coping item bank had 30 items. Rasch analysis was used to assess the psychometric properties. A CAT simulation was carried out to estimate an average number of items required to gain precise measurement of hereditary retinal disease-related coping. RESULTS: One hundred eighty-nine participants answered the coping item bank (median age = 58 years). The coping scale demonstrated good precision and targeting. The standardised residual loadings for items revealed six items grouped together. Removal of the six items reduced the precision of the main coping scale and worsened the variance explained by the measure. Therefore, the six items were retained within the main scale. Our CAT simulation indicated that, on average, less than 10 items are required to gain a precise measurement of coping. CONCLUSIONS: This is the first study to develop a psychometrically robust coping instrument for hereditary retinal diseases. CAT simulation indicated that on an average, only four and nine items were required to gain measurement at moderate and high precision, respectively.

Genetic analysis of choroideremia families in the Australian population.

BACKGROUND: Choroideremia is an X-linked inherited chorioretinal disease known to be caused by mutations in the CHM gene. In this study, Australian families clinically diagnosed with choroideremia were genetically analysed for mutations in the CHM gene. DESIGN: The Australian Inherited Retinal Disease Register and DNA Bank (AIRDR) was investigated to identify a cohort of choroideremia-affected families for genetic analysis. PARTICIPANTS: Participants were sourced from the AIRDR. Thirty-two participants (15 affected, 10 carriers, 7 unaffected) sourced from 11 unrelated families having at least one member clinically diagnosed with choroideremia were included in the study. METHODS: We performed sequence analysis of the CHM gene on the DNA of nine probands. We received the direct sequencing results of two probands by other means. Targeted analysis was subsequently performed for all 32 participants to confirm the direct sequencing results in the 11 probands and to establish the presence or absence of the implicated mutation in the remaining 21 affected, carrier or unaffected family members. MAIN OUTCOME MEASURES: Genetic characterisation of 11 choroideremia families in the Australian population. RESULTS: A CHM mutation was detected in all 11 families. Each family had a different mutation. Mutations segregated within each family according to disease status. Five mutations were novel and six have been previously reported. CONCLUSIONS: Six previously reported and five novel CHM mutations were detected in 11 Australian families clinically diagnosed with choroideremia. We anticipate that this work will facilitate access for AIRDR participants and their progeny to CHM gene therapy trials.

Clinical and molecular characterization of females affected by X-linked retinoschisis.

BACKGROUND: X-linked retinoschisis (XLRS) is a leading cause of juvenile macular degeneration associated with mutations in the RS1 gene. XLRS has a variable expressivity in males and shows no clinical phenotype in carrier females. DESIGN: Clinical and molecular characterization of male and female individuals affected with XLRS in a consanguineous family. PARTICIPANTS: Consanguineous Eastern European-Australian family METHODS: Four clinically affected and nine unaffected family members were genetically and clinically characterized. Deoxyribonucleic acid (DNA) analysis was conducted by the Australian Inherited Retinal Disease Register and DNA Bank. MAIN OUTCOME MEASURES: Clinical and molecular characterization of the causative mutation in a consanguineous family with XLRS. RESULTS: By direct sequencing of the RS1 gene, one pathogenic variant, NM_000330.3: c.304C > T, p. R102W, was identified in all clinically diagnosed individuals analysed. The two females were homozygous for the variant, and the males were hemizygous. CONCLUSION: Clinical and genetic characterization of affected homozygous females in XLRS affords the rare opportunity to explore the molecular mechanisms of XLRS and the manifestation of these mutations as disease in humans.

Censoring the Floor Effect in Long-Term Stargardt Disease Microperimetry Data Produces a Faster Rate of Decline.

Purpose: To evaluate progression rate estimation in long-term Stargardt disease microperimetry data by accounting for floor effect. Design: Cohort study. Subjects: Thirty-seven subjects (23 females, 14 males) with biallelic ABCA4 pathogenic or likely pathogenic variants and more than >2 years of longitudinal microperimetry data. Methods: Cross-sectional and longitudinal microperimetry data (Grid A: 18° diameter, Grid B: 6° diameter; Macular Integrity Assessment microperimeter, dynamic range 0-36 decibels [dB]) was extracted from patients with biallelic mutation in the adenosine triphosphate-binding cassette subfamily A member 4 (ABCA4) gene. For each eye, mean sensitivity (MS) and responding point sensitivity (RPS) rates were extracted. Floor censored sensitivity (FCS) progression rate, which accounts for the floor effect at each locus by terminating calculation when scotoma was observed in 2 consecutive visits, was also calculated. In a subset of eyes with ≥1 scotomatous locus at baseline (Grid A), sensitivity progression of loci around the scotoma (edge of scotoma sensitivity [ESS]) was examined against other progression parameters. Paired t test compared progression rate parameters across the same eyes. Main Outcome Measures: Microperimetry grid parameters at baseline and progression rates. Results: A total of 37 subjects with biallelic ABCA4 mutations and >2 years of longitudinal microperimetry data were included in the study. In Grid A, at baseline, the average MS and RPS were 16.5 ± 7.9 and 19.1 ± 5.7 dB, respectively. Similar MS (18.4 ± 7.6 dB) and RPS (20.0 ± 5.5 dB) values were found at baseline for Grid B. In Grid A, overall, MS, RPS, and FCS progression rates were -0.57 ± 1.05, -0.74 ± 1.24, and -1.26 ± 1.65 (all dB/year), respectively. Floor censored sensitivity progression rate was significantly greater than the MS or RPS progression rates. Similar findings were observed in Grid B (MS -1.22 ± 1.42, RPS -1.44 ± 1.44, FCS -2.16 ± 2.24, all dB/year), with paired t test again demonstrated that FCS had a significantly faster rate of decline than MS or RPS. In patients with progression data in both grids, MS, RPS, and FCS progression rates were significantly faster in the smaller Grid B. In 24 eyes with scotoma at baseline, fastest rate of decline was ESS combined with FCS compared with other progression parameters. Conclusions: Incorporation of FCS can reduce confound of floor effect in perimetry analysis and can in turn detect a faster rate of decline. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.

Establishment of an induced pluripotent stem cell line LEIi019-A from an early-onset retinal dystrophy patient with the autosomal dominant OTX2 c.259G>A variant.

The human induced pluripotent stem cell (iPSC) line LEIi019-A was generated from a patient with early-onset pattern dystrophy caused by a heterozygous mutation NM_001270525.1:c.259G>A (p.Glu87Lys) in OTX2. Patient-derived dermal fibroblasts were reprogrammed using episomal plasmids containing reprogramming factors OCT4, SOX2, KLF4, MYCL, LIN28, TP53 shRNA and miR-302/367. The iPSC line expressed pluripotency markers, displayed a normal 46,XY karyotype and demonstrated the ability to differentiate into the three primary germ layers, retinal organoids and retinal pigment epithelial cells.

Analysis of the Outer Retinal Bands in ABCA4 and PRPH2-Associated Retinopathy using OCT.

PURPOSE: To evaluate the outer retinal bands using OCT in ABCA4- and PRPH2-associated retinopathy and develop a novel imaging biomarker to differentiate between these 2 genotypes. DESIGN: Multicenter case-control study. PARTICIPANTS: Patients with a clinical and genetic diagnosis of ABCA4- or PRPH2-associated retinopathy and an age-matched control group. METHODS: Macular OCT was used to measure the thickness of the outer retinal bands 2 and 4 by 2 independent examiners at 4 retinal loci. MAIN OUTCOME MEASURES: Outcome measures included the thicknesses of band 2, band 4, and the band 2/band 4 ratio. Linear mixed modeling was used to make comparisons across the 3 groups. Receiver operating characteristic (ROC) analysis determined the optimal cutoff for the band 2/band 4 ratio to distinguish PRPH2- from ABCA4-associated retinopathy. RESULTS: We included 45 patients with ABCA4 variants, 45 patients with PRPH2 variants, and 45 healthy controls. Band 2 was significantly thicker in patients with PRPH2 compared with ABCA4 (21.4 vs. 15.9 µm, P < 0.001) variants, whereas band 4 was thicker in patients with ABCA4 variants than those with PRPH2 variants (27.5 vs. 21.7 µm, P < 0.001). Similarly, the band 2/band 4 ratio was significantly different (1.0 vs. 0.6 for PRPH2 vs. ABCA4, P < 0.001). The area under the ROC curve was 0.87 for either band 2 (> 18.58 µm) or band 4 (< 26.17 µm) alone and 0.99 (95% confidence interval: 0.97-0.99) for the band 2/band 4 ratio with a cutoff threshold of 0.79, providing 100% specificity. CONCLUSIONS: We report an altered outer retinal band profile whereby the band 2/band 4 ratio was able to discriminate between PRPH2- and ABCA4-associated retinopathy. This may have future clinic utility in predicting the genotype and provide further insight into the anatomic correlate of band 2. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Generation of two induced pluripotent stem cell lines from an Usher syndrome type 1B patient with the homozygous c.496del MYO7A variant.

Usher syndrome (USH) is the most common cause of inherited deaf-blindness. Here, we produced the LEIi020-A and LEIi020-B induced pluripotent stem cell (iPSC) lines from dermal fibroblasts derived from a patient with USH1B caused by inheritance of homozygous c.496del variants in MYO7A using episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for TP53. Both iPSC lines expressed pluripotency markers, demonstrated trilineage differentiation potential and displayed a 46,XY karyotype. These cell lines represent a valuable resource for the production of retinal and otic tissues to support research into the pathogenesis and treatment of USH1B.

Retinal Characteristics of Female Choroideremia Carriers: Multimodal Imaging, Microperimetry, and Genetics.

PURPOSE: To describe visual function and retinal features of female carriers of choroideremia (CHM), using multimodal imaging and microperimetry. DESIGN: Cross-sectional cohort study. PARTICIPANTS AND CONTROLS: Choroideremia carriers seen in Australia (Melbourne or Perth) or the United Kingdom (Oxford or Cambridge) between 2012 and 2023. Healthy age-matched controls seen in Melbourne, Australia, between 2022 and 2023. METHODS: Participants had visual acuity, fundus-tracked microperimetry, OCT, and fundus autofluorescence imaging performed. Choroideremia carriers were either genetically or clinically confirmed (i.e., obligate carriers). Choroideremia carriers were grouped according to their retinal phenotype and compared with healthy controls. Statistical analyses were performed on StataBE (v18.0). MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA), low-luminance visual acuity (LLVA), average retinal sensitivity, volume of macular hill of vision (HoV), inner retinal thickness, and photoreceptor complex (PRC) thickness. RESULTS: Eighty-six eyes of 43 CHM carriers and 60 eyes of 30 healthy controls were examined using multimodal imaging and microperimetry. Median age was 54 and 48.5 years for CHM carriers and controls, respectively (P = 0.18). Most CHM carriers (86%) were genetically confirmed. Choroideremia carriers and controls had strong intereye correlation between eyes for BCVA and average retinal sensitivity (P < 0.001). Low-luminance visual acuity and macular HoV tests were sensitive tests to detect changes in CHM carriers with mild phenotypes (i.e., fine and coarse). Choroideremia carriers with geographic or male-pattern phenotypes had reduced BCVA, LLVA, retinal sensitivity, and retinal thinning, compared with healthy controls. Retinal thickening of the inner retina was observed in the central 1°, despite generalized thinning of the PRC in the central 7°, indicating retinal remodeling in CHM carriers, compared with controls. There were no genotype-phenotype correlations observed. CONCLUSIONS: Female carriers of CHM with severe retinal phenotypes (i.e., geographic or male pattern) have significantly decreased visual function and retinal structural changes when compared with age-matched controls and those carriers with milder phenotypes. Low-luminance visual acuity and volumetric measures of the macular HoV were found to be the most sensitive functional tests to detect milder retinal disease (fine and coarse phenotypes) in CHM carriers. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum and Novel Clinical Observations in 241 Patients.

Purpose: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene. Methods: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations. Results: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, or vitelliform macular dystrophy (11%), central areolar choroidal dystrophy (28%), pseudo-Stargardt pattern dystrophy (41%), and retinitis pigmentosa (25%). Symptom onset was earlier in retinitis pigmentosa as compared with pseudo-Stargardt pattern dystrophy (34 vs 44 years; P = 0.004). The median visual acuity was 0.18 logMAR (interquartile range, 0-0.54 logMAR) and 0.18 logMAR (interquartile range 0-0.42 logMAR) in the right and left eyes, respectively. ERG showed a significantly reduced amplitude across all components (P < 0.001) and a peak time delay in the light-adapted 30-Hz flicker and single-flash b-wave (P < 0.001). Twenty-two variants were novel. The central areolar choroidal dystrophy phenotype was associated with 13 missense variants. The remaining variants showed marked phenotypic variability. Conclusions: We described six distinct FAF phenotypes associated with variants in the PRPH2 gene. One FAF phenotype may have multiple ERG phenotypes, demonstrating a discordance between structure and function. Given the vast spectrum of PRPH2 disease our findings are useful for future clinical trials.