Biallelic Loss-of-Function Variants in UBAP1L and Nonsyndromic Retinal Dystrophies.

Importance: Inherited retinal dystrophies (IRDs) present a challenge in clinical diagnostics due to their pronounced genetic heterogeneity. Despite advances in next-generation sequencing (NGS) technologies, a substantial portion of the genetic basis underlying IRDs remains elusive. Addressing this gap seems important for gaining insights into the genetic landscape of IRDs, which may help improve diagnosis and prognosis and develop targeted therapies in the future. Objective: To provide a clinical and molecular characterization of 6 patients with IRDs with biallelic disease-causing variants in a novel candidate IRD disease gene. Design, Setting, and Participants: This multicenter case series study included 6 patients with IRDs from 4 tertiary hospitals (in the US: National Eye Institute, National Institutes of Health Clinical Center; in the UK: Moorfields Eye Hospital, Royal Liverpool University Hospital, Birmingham Women's and Children's). Exposures: Biallelic disease-causing variants in the novel candidate IRD disease gene, UBAP1L. Main Outcome and Measures: Participants underwent comprehensive clinical ophthalmic assessments to characterize the features of retinal dystrophy. Exome and genome sequencing revealed candidate variants in the UBAP1L gene; no other plausible disease variants in known IRD genes were identified. A minigene assay provided functional insights for a noncanonical splice variant, and a knockout mouse model was used for in vivo functional elucidation. Results: Four homozygous UBAP1L variants were identified in the affected individuals from 6 families, including 2 frameshift variants (c.710del and c.634_644del), 1 canonical splice variant (c.121-2A>C), and 1 noncanonical splice variant (c.910-7G>A), which was shown to cause aberrant splicing and frameshift in a minigene assay. Participants presented with retinal dystrophy including maculopathy, cone dystrophy, and cone-rod dystrophy. Single-cell RNA sequencing of the retina showed that human UBAP1L is highly expressed in both cones and retinal pigment epithelium, whereas mouse Ubap1l is highly expressed in cone cells only. Mice with truncation of the C-terminal SOUBA domain did not manifest retinal degeneration up to 15 months of age. Conclusions and Relevance: Study results reveal clinical and genetic evidence that loss of UBAP1L function was associated with inherited retinopathy in humans. These findings hold promise for improved clinical diagnostics, prognosis, and the potential development of targeted therapies for individuals affected by IRDs.

PDE6A-Associated Retinitis Pigmentosa, Clinical Characteristics, Genetics and Natural History.

PURPOSE: To analyze the genetics, clinical characteristics, and natural history of PDE6A-associated retinitis pigmentosa. DESIGN: Retrospective, longitudinal, observational cohort study. PARTICIPANTS: Patients with molecularly confirmed PDE6A-associated retinal dystrophy in a single tertiary referral center. METHODS: Review of medical records and retinal imaging, including fundus autofluorescence (FAF) imaging and spectral-domain optical coherence tomography (SD-OCT). Genetic results were reviewed, and the detected variants were assessed. RESULTS: Sixteen patients (32 eyes) were identified and evaluated longitudinally. Genetic analysis identified 14 variants in the PDE6A gene, including 8 novel variants. The mean age (±SD, range) was 34.8 years (± 17.4, 12 - 76) at baseline, with a mean follow-up time of 4.8 years. Best-corrected visual acuity (BCVA) was 0.45 ± 0.45 LogMAR (range 0.0 - 1.6) at baseline and 0.65 ± 0.7 LogMAR (range 0.0 - 2.3) at the last visit. BCVA was similar among eyes in 88% of patients. A hyperautofluorescent ring was observed on FAF in 50% and 44% of the eyes at baseline and follow up visit respectively, with a mean area of 9.7 ± 4.5mm2 at baseline and mean of 8.6 ± 4.8 mm2 at the follow-up visit. Mean horizontal ellipsoid zone width (EZW) at baseline was 1765 ± 1093 µm, which decreased to 1580 ± 1077 µm at follow up. Eighteen eyes exhibited cystoid macular oedema at baseline (56%), and 17 eyes (53%) at follow-up. There were statistically significant changes during the follow-up period in terms of BCVA, hyperautoflouroscent ring area and the EZW. CONCLUSIONS: This study highlights the natural history of PDE6A-retinopathy. The majority of the patients in this cohort had mild BCVA loss, and slowly progressive disease, based on FAF and OCT measurements.

Bi-allelic variants in COQ8B, a gene involved in the biosynthesis of coenzyme Q10, lead to non-syndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is a Mendelian disease characterized by gradual loss of vision, due to the progressive degeneration of retinal cells. Genetically, it is highly heterogeneous, with pathogenic variants identified in more than 100 genes so far. Following a large-scale sequencing screening, we identified five individuals (four families) with recessive and non-syndromic RP, carrying as well bi-allelic DNA changes in COQ8B, a gene involved in the biosynthesis of coenzyme Q10. Specifically, we detected compound heterozygous assortments of five disease-causing variants (c.187C>T [p.Arg63Trp], c.566G>A [p.Trp189Ter], c.1156G>A [p.Asp386Asn], c.1324G>A [p.Val442Met], and c.1560G>A [p.Trp520Ter]), all segregating with disease according to a recessive pattern of inheritance. Cell-based analysis of recombinant proteins deriving from these genotypes, performed by target engagement assays, showed in all cases a significant decrease in ligand-protein interaction compared to the wild type. Our results indicate that variants in COQ8B lead to recessive non-syndromic RP, possibly by impairing the biosynthesis of coenzyme Q10, a key component of oxidative phosphorylation in the mitochondria.

Investigating Splice Defects in USH2A Using Targeted Long-Read Sequencing.

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

Outer Retinal Columnar Abnormalities (ORCA): a novel optical coherence tomography sign of CRB1 maculopathy?

PURPOSE: To report a novel optical coherence tomography (OCT) sign in the context of CRB1-related maculopathy, termed outer retinal columnar abnormalities (ORCA). METHODS: Retrospective, multicenter observational case series of 14 eyes of 8 patients with molecularly confirmed CRB1-related maculopathy and ORCA. Multimodal imaging scans and medical records patients with CRB1-related maculopathy were reviewed. Outcome measures included best-corrected visual acuity (BCVA), central subfield thickness on spectral-domain OCT (SD-OCT), presence of ORCAs and analysis of their change in appearance over time. RESULTS: At baseline, mean age was 18 +/- 10 years (range 9-36). All patients had an isolated macular dystrophy except for 1 case harboring a triallelic pathogenic variant. Variant c.498_506del was found in 9 cases (88%). At presentation, ORCA were visible on macular SD-OCT in all cases as multiform, vertical hyperreflective columnar alterations extending from the ellipsoid to the outer plexiform layer, with a variable degree of hyporeflective cystic spaces in the outer and inner nuclear layers. Over 6 +/- 4.7 follow-up years, the presence of ORCA varied greatly with a decrease in ORCA associated with sequential development of retinal atrophy. CONCLUSIONS: A high suspicion for CRB1-associated retinal dystrophy should arise in the presence of ORCA on SD-OCT, prompting genetic testing.

Mild retinitis pigmentosa, including sector retinitis pigmentosa associated with 2 pathogenic variants in CDH23.

BACKGROUND: Biallelic pathogenic variants in CDH23 can cause Usher syndrome type I (USH1), typically characterized by sensorineural hearing loss, variable vestibular areflexia, and a progressive form of rod-cone dystrophy. While missense variants in CDH23 can cause DFNB12 deafness, other variants can affect the cadherin 23 function, more severely causing Usher syndrome type I D. The main purpose of our study is to describe the genotypes and phenotypes of patients with mild retinitis pigmentosa (RP), including sector RP with two pathogenic variants in CDH23. MATERIALS AND METHODS: Clinical examination included medical history, comprehensive ophthalmologic examination, and multimodal retinal imaging, and in case 1 and 2, full-field electroretinography (ERG). Genetic analysis was performed in all cases, and segregation testing of proband relatives was performed in case 1 and 3. RESULTS: Three unrelated cases presented with variable clinical phenotype for USH1 and were found to have two pathogenic variants in CDH23, with missense variant, c.5237 G > A: p.Arg1746Gln being common to all. All probands had mild to profound hearing loss. Case 1 and 3 had mild RP with mid peripheral and posterior pole sparing, while case 2 had sector RP. ERG results were consistent with the marked loss of retinal function in both eyes at the level of photoreceptor in case 1 and case 2, with normal peak time in the former. CONCLUSION: Patients harbouring c.5237 G > A: p.Arg1746Gln variants in CDH23 can present with a mild phenotype including sector RP. This can aid in better genetic counselling and in prognostication.

Clinical, Genetic and Histopathological Characteristics of CRX-associated Retinal Dystrophies.

PURPOSE: To describe phenotypic, genotypic, and histopathological features of inherited retinal dystrophies associated with the CRX gene (CRX-RDs). DESIGN: Retrospective multicenter cohort study including histopathology. SUBJECTS: Thirty-nine patients from 31 families with pathogenic variants in the CRX gene. METHODS: Clinical data of 152 visits were collected from medical records with a median follow-up time of 9.1 years (IQR, 3.3-15.3 years; range, 0.0-48.8 years). Histopathologic examination of the eye of a 17-year-old patient with advanced early-onset CRX-RD was performed. MAIN OUTCOME MEASURES: Visual acuity, retinal imaging, electroretinography (ERG), genotype-phenotype correlation, and histopathological examination were evaluated. RESULTS: The age at onset ranged from birth to the 8th decade of life. Median visual acuity was 1.00 logMAR (IQR, 0.69-1.48 logMAR; range, 0.06-3.00 logMAR) at a mean age of 52.0 ± 19.9 years (range, 4.6-81.9 years). Sufficient imaging was available for 36 out of 39 patients (92.3%), and all showed degeneration of at least the macula. Out of these 36 patients, 22 (61.1%) had only macular dystrophy. Another 10 patients (27.8%) had additional degeneration beyond the vascular arcades, and 4 patients (11.1%) panretinal degeneration. Two patients (5.1%) had Leber congenital amaurosis (LCA). In total, 21 different disease-associated heterozygous CRX variants were identified (10 missense, 8 frameshift, 2 deletion, 1 deletion-insertion variants). Missense variants in the CRX homeodomain and two variants deleting all functional domains, thus causing haploinsufficiency, generally tended to cause milder late-onset phenotypes. Histopathologic examination of the eye of a 17-year-old patient with advanced early-onset retinal dystrophy due to a heterozygous deletion of exons 3 and 4 of the CRX gene revealed loss of laminar integrity and widespread photoreceptor degeneration especially in the central retina, with extensive loss of photoreceptor nuclei and outer segments. CONCLUSION: This study illustrates the large clinical and genetic heterogenic spectrum of CRX-RDs, ranging from LCA to mild late-onset maculopathy resembling occult macular dystrophy. Haploinsufficiency and missense variants tended to be associated with milder phenotypes. Patients showed degeneration predominantly affecting the central retina on imaging. The histopathological findings also mirror these clinical findings and features similar to previously reported animal models of CRX-RDs.

Substitution of a single non-coding nucleotide upstream of TMEM216 causes non-syndromic retinitis pigmentosa and is associated with reduced TMEM216 expression.

Genome analysis of individuals affected by retinitis pigmentosa (RP) identified two rare nucleotide substitutions at the same genomic location on chromosome 11 (g.61392563 [GRCh38]), 69 base pairs upstream of the start codon of the ciliopathy gene TMEM216 (c.-69G>A, c.-69G>T [GenBank: NM_001173991.3]), in individuals of South Asian and African ancestry, respectively. Genotypes included 71 homozygotes and 3 mixed heterozygotes in trans with a predicted loss-of-function allele. Haplotype analysis showed single-nucleotide variants (SNVs) common across families, suggesting ancestral alleles within the two distinct ethnic populations. Clinical phenotype analysis of 62 available individuals from 49 families indicated a similar clinical presentation with night blindness in the first decade and progressive peripheral field loss thereafter. No evident systemic ciliopathy features were noted. Functional characterization of these variants by luciferase reporter gene assay showed reduced promotor activity. Nanopore sequencing confirmed the lower transcription of the TMEM216 c.-69G>T allele in blood-derived RNA from a heterozygous carrier, and reduced expression was further recapitulated by qPCR, using both leukocytes-derived RNA of c.-69G>T homozygotes and total RNA from genome-edited hTERT-RPE1 cells carrying homozygous TMEM216 c.-69G>A. In conclusion, these variants explain a significant proportion of unsolved cases, specifically in individuals of African ancestry, suggesting that reduced TMEM216 expression might lead to abnormal ciliogenesis and photoreceptor degeneration.

CDH23-Associated Usher Syndrome: Clinical Features, Retinal Imaging, and Natural History.

Purpose: The purpose of this study was to analyze the clinical spectrum and natural history of CDH23-associated Usher syndrome type ID (USH1D). Methods: Molecularly-confirmed individuals had data extracted from medical records. Retinal imaging was extracted from an in-house database. The main outcome measurements were retinal imaging and electroretinography (ERG) and clinical findings, including age of onset, symptoms, best-corrected visual acuity (BCVA), outer nuclear layer (ONL) thickness, ellipsoid zone width (EZW), and hyperautofluorescent ring area. Results: Thirty-one patients were identified, harboring 40 variants in CDH23 (10 being novel). The mean (range, ±SD) age of symptom onset was 10.1 years (range = 1-18, SD = ±4.1). The most common visual symptoms at presentation were nyctalopia (93.5%) and peripheral vision difficulties (61.3%). The mean BCVA at baseline was 0.25 ± 0.22 in the right eyes and 0.35 ± 0.58 LogMAR in the left eyes. The mean annual loss rate in BCVA was 0.018 LogMAR/year over a mean follow-up of 9.5 years. Individuals harboring the c.5237G>A p.(Arg1746Gln) allele had retinitis pigmentosa (RP) sparing the superior retina. Seventy-seven percent of patients had hyperautofluorescent rings in fundus autofluorescence. Full-field and pattern ERGs indicated moderate-severe rod-cone or photoreceptor dysfunction with relative sparing of macular function in most patients tested. Optical coherence tomography (OCT) revealed intraretinal cysts in the transfoveal B-scan of 13 individuals (43.3%). The rate of EZW and ONL thickness loss was mild and suggestive of a wide window of macular preservation. Conclusions: Despite the early onset of symptoms, USH1D has a slowly progressive phenotype. There is high interocular symmetry across all parameters, making it an attractive target for novel therapies.

Characterising the refractive error in paediatric patients with congenital stationary night blindness: a multicentre study.

BACKGROUND/AAIMS: Congenital stationary night blindness (CSNB) is an inherited retinal disease that is often associated with high myopia and can be caused by pathological variants in multiple genes, most commonly CACNA1F, NYX and TRPM1. High myopia is associated with retinal degeneration and increased risk for retinal detachment. Slowing the progression of myopia in patients with CSNB would likely be beneficial in reducing risk, but before interventions can be considered, it is important to understand the natural history of myopic progression. METHODS: This multicentre, retrospective study explored CSNB caused by variants in CACNA1F, NYX or TRPM1 in patients who had at least 6 measurements of their spherical equivalent of refraction (SER) before the age of 18. A mixed-effect model was used to predict progression of SER overtime and differences between genotypes were evaluated. RESULTS: 78 individuals were included in this study. All genotypes showed a significant myopic predicted SER at birth (-3.076D, -5.511D and -5.386D) for CACNA1F, NYX and TRPM1 respectively. Additionally, significant progression of myopia per year (-0.254D, -0.257D and -0.326D) was observed for all three genotypes CACNA1F, NYX and TRPM1, respectively. CONCLUSIONS: Patients with CSNB tend to be myopic from an early age and progress to become more myopic with age. Patients may benefit from long-term myopia slowing treatment in the future and further studies are indicated. Additionally, CSNB should be considered in the differential diagnosis for early-onset myopia.

Enhanced Learning and Memory in Patients with CRB1 Retinopathy.

Mutations in the CRB1 gene are associated with a diverse spectrum of retinopathies with phenotypic variability causing severe visual impairment. The CRB1 gene has a role in retinal development and is expressed in the cerebral cortex and hippocampus, but its role in cognition has not been described before. This study compares cognitive function in CRB1 retinopathy individuals with subjects with other retinopathies and the normal population. METHODS: Neuropsychological tests of cognitive function were used to test individuals with CRB1 and non-CRB1 retinopathies and compare results with a standardised normative dataset. RESULTS: CRB1 retinopathy subjects significantly outperformed those with non-CRB1 retinopathy in list learning tasks of immediate (p = 0.001) and delayed memory (p = 0.007), tests of semantic verbal fluency (p = 0.017), verbal IQ digit span subtest (p = 0.037), and estimation test of higher execution function (p = 0.020) but not in the remaining tests of cognitive function (p > 0.05). CRB1 retinopathy subjects scored significantly higher than the normal population in all areas of memory testing (p < 0.05) and overall verbal IQ tests (p = 0.0012). Non-CRB1 retinopathy subjects scored significantly higher than the normal population in story recall, verbal fluency, and overall verbal IQ tests (p = 0.0016). CONCLUSIONS: Subjects with CRB1 retinopathy may have enhanced cognitive function in areas of memory and learning. Further work is required to understand the role of CRB1 in cognition.

A Long-Term Retrospective Natural History Study of EFEMP1-Associated Autosomal Dominant Drusen.

Purpose: To analyze the natural history of EFEMP1-associated autosomal dominant drusen (ADD). Methods: In this retrospective observational study of molecularly confirmed patients with ADD, data and retinal imaging were extracted from an in-house database. The main outcome measurements were best-corrected visual acuity (BCVA), refraction, and retinal imaging, including quantitative analyses of the outer nuclear layer (ONL) thickness and pigmented epithelium detachment area, as well as qualitative analyses. Results: The study included 44 patients (34 females and 10 males). The mean ± SD age of symptom onset was 40.1 ± 6.59 years of age (range, 25-52). Fourteen patients were asymptomatic during their entire follow-up. The most common symptoms at presentation were reduced vision (70%) and distortion in central vision (53%). Most subjects were emmetropic. The mean BCVA (logMAR) at baseline was 0.27 ± 0.41 (range, -0.1 to 2.1) in right eyes and was 0.19 ± 0.32 (range, -0.2 to 1.3) in left eyes. After a mean follow-up of 7.9 years, BCVA was reduced to 0.59 ± 0.66 (range, -0.1 to 2.1) in right eyes and 0.5 ± 0.72 (range, -0.1 to 2.4) in left eyes, values that were significantly different than baseline (P < 0.0001 and P < 0.0014, respectively). Fifteen eyes showed active or inactive choroidal neovascularization (CNV). BCVA differed significantly (P = 0.0004) between eyes with and without CNV at a comparable mean age. The ONL had a slow rate of thinning longitudinally, which significantly correlated with BCVA. Conclusions: Despite the late onset and relatively good prognosis of ADD, CNVs are more frequent than previously reported and are associated with a worse prognosis. Further research is necessary to elucidate gender associations.

Non-syndromic retinal dystrophy associated with biallelic variation of SUMF1 and reduced leukocyte sulfatase activity.

Biallelic variants in SUMF1 are associated with multiple sulfatase deficiency (MSD), a rare lysosomal storage disorder typically diagnosed in early infancy or childhood, marked by severe neurodegeneration and early mortality. We present clinical and molecular characterisation of three unrelated patients aged 13 to 58 years with milder clinical manifestations due to SUMF1 disease variants, including two adult patients presenting with apparent non-syndromic retinal dystrophy. Whole genome sequencing identified biallelic SUMF1 variants in all three patients; Patient 1 homozygous for a complex allele c.[290G>T;293T>A]; p.[(Gly97Val);(Val98Glu)], Patient 2 homozygous for c.866A>G; p.(Tyr289Cys), and Patient 3 compound heterozygous for c.726-1G>C and p.(Tyr289Cys). Electroretinography indicated a rod-cone dystrophy with additional possible inner retinal dysfunction in all three patients. Biochemical studies confirmed reduced, but not absent, sulfatase enzyme activity in the absence of extra-ocular disease (Patient 1) or only mild systemic disease (Patients 2, 3). These cases are suggestive that non-null SUMF1 genotypes can cause an attenuated clinical phenotype, including retinal dystrophy without systemic complications, in adulthood.

Clinical, Ophthalmic, and Genetic Characterization of RPGRIP1-Associated Leber Congenital Amaurosis/Early-Onset Severe Retinal Dystrophy.

PURPOSE: To present the clinical characteristics, retinal features, natural history, and genetics of RPGRIP1-associated early-onset severe retinal dystrophy (EOSRD)/Leber congenital amaurosis (LCA). DESIGN: Retrospective case series. METHODS: Review of clinical notes, multimodal retinal imaging, and molecular diagnosis of 18 patients (17 families) with EOSRD/LCA and disease-causing variants in RPGRIP1. RESULTS: The mean age of visual symptoms onset was 0.87 ± 1 year (birth to 3 years), and the mean age at baseline visit was 11.4 ± 10.2 years (1-39 years). At the baseline visit, 44% of patients were legally blind (range, 2-39 years), and there was no significant association found between age and best-corrected visual acuity (BCVA) in cross-sectional analysis. Retinal evaluation showed an abolished electroretinogram or a cone-rod dystrophy pattern, no or minimal pigment deposits, a hyperautofluorescent ring at the posterior pole, and a largely preserved central macular architecture, with retained outer nuclear layer and ellipsoid zone island into adulthood. Eleven variants (48%) were previously unreported, and 13 families (76%) had a double-null (DN) genotype. Twelve patients (67%) had follow-up assessments over a 15.7 ± 9.5-year period. The rate of BCVA decline was 0.02 logarithm of the minimum angle of resolution (1 letter)/year. CONCLUSIONS: RPGRIP1 EOSRD/LCA often presents at birth or early infancy, with nystagmus, decreased visual acuity, hyperopia, and photophobia. Patients with a DN genotype may develop symptoms earlier and have worse vision. Multimodal imaging may show a hyperautofluorescent posterior pole ring and relatively preserved central macular architecture, suggesting that the condition is a promising candidate for gene supplementation.

Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

Sex Distributions in Non-ABCA4 Autosomal Macular Dystrophies.

Purpose: We sought to explore whether sex imbalances are discernible in several autosomally inherited macular dystrophies. Methods: We searched the electronic patient records of our large inherited retinal disease cohort, quantifying numbers of males and females with the more common (non-ABCA4) inherited macular dystrophies (associated with BEST1, EFEMP1, PROM1, PRPH2, RP1L1, and TIMP3). BEST1 cases were subdivided into typical autosomal dominant and recessive disease. For PRPH2, only patients with variants at codons 172 or 142 were included. Recessive PROM1 and recessive RP1L1 cases were excluded because these variants give a more widespread or peripheral degeneration. The proportion of females was calculated for each condition; two-tailed binomial testing was performed. Where a significant imbalance was found, previously published cohorts were also explored. Results: Of 325 patients included, numbers for BEST1, EFEMP1, PROM1, PRPH2, RP1L1, and TIMP3 were 152, 35, 30, 50, 14, and 44, respectively. For autosomal dominant Best disease (n = 115), there were fewer females (38%; 95% confidence interval [CI], 29-48%; P = 0.015). For EFEMP1-associated disease (n = 35), there were significantly more females (77%; 95% CI, 60%-90%; P = 0.0019). No significant imbalances were seen for the other genes. When pooling our cohort with previous large dominant Best disease cohorts, the proportion of females was 37% (95% CI, 31%-43%; P = 1.2 × 10-5). Pooling previously published EFEMP1-cases with ours yielded an overall female proportion of 62% (95% CI, 54%-69%; P = 0.0023). Conclusions: This exploratory study found significant sex imbalances in two autosomal macular dystrophies, suggesting that sex could be a modifier. Our findings invite replication in further cohorts and the investigation of potential mechanisms.

Quantification of Fundus Autofluorescence Features in a Molecularly Characterized Cohort of More Than 3500 Inherited Retinal Disease Patients from the United Kingdom.

Purpose: To quantify relevant fundus autofluorescence (FAF) image features cross-sectionally and longitudinally in a large cohort of inherited retinal diseases (IRDs) patients. Design: Retrospective study of imaging data (55-degree blue-FAF on Heidelberg Spectralis) from patients. Participants: Patients with a clinical and molecularly confirmed diagnosis of IRD who have undergone FAF 55-degree imaging at Moorfields Eye Hospital (MEH) and the Royal Liverpool Hospital (RLH) between 2004 and 2019. Methods: Five FAF features of interest were defined: vessels, optic disc, perimacular ring of increased signal (ring), relative hypo-autofluorescence (hypo-AF) and hyper-autofluorescence (hyper-AF). Features were manually annotated by six graders in a subset of patients based on a defined grading protocol to produce segmentation masks to train an AI model, AIRDetect, which was then applied to the entire MEH imaging dataset. Main Outcome Measures: Quantitative FAF imaging features including area in mm 2 and vessel metrics, were analysed cross-sectionally by gene and age, and longitudinally to determine rate of progression. AIRDetect feature segmentation and detection were validated with Dice score and precision/recall, respectively. Results: A total of 45,749 FAF images from 3,606 IRD patients from MEH covering 170 genes were automatically segmented using AIRDetect. Model-grader Dice scores for disc, hypo-AF, hyper-AF, ring and vessels were respectively 0.86, 0.72, 0.69, 0.68 and 0.65. The five genes with the largest hypo-AF areas were CHM , ABCC6 , ABCA4 , RDH12 , and RPE65 , with mean per-patient areas of 41.5, 30.0, 21.9, 21.4, and 15.1 mm 2 . The five genes with the largest hyper-AF areas were BEST1 , CDH23 , RDH12 , MYO7A , and NR2E3 , with mean areas of 0.49, 0.45, 0.44, 0.39, and 0.34 mm 2 respectively. The five genes with largest ring areas were CDH23 , NR2E3 , CRX , EYS and MYO7A, with mean areas of 3.63, 3.32, 2.84, 2.39, and 2.16 mm 2 . Vessel density was found to be highest in EFEMP1 , BEST1 , TIMP3 , RS1 , and PRPH2 (10.6%, 10.3%, 9.8%, 9.7%, 8.9%) and was lower in Retinitis Pigmentosa (RP) and Leber Congenital Amaurosis genes. Longitudinal analysis of decreasing ring area in four RP genes ( RPGR, USH2A, RHO, EYS ) found EYS to be the fastest progressor at -0.18 mm 2 /year. Conclusions: We have conducted the first large-scale cross-sectional and longitudinal quantitative analysis of FAF features across a diverse range of IRDs using a novel AI approach.