Michel Michaelides

The Natural History of the Progression of Atrophy Secondary to Stargardt Disease (ProgStar) Studies: Design and Baseline Characteristics: ProgStar Report No. 1

PURPOSE To describe the design and baseline characteristics of patients enrolled into 2 natural history studies of Stargardt disease (STGD1). DESIGN Multicenter retrospective and prospective cohort studies. PARTICIPANTS Three hundred sixty-five unique patients aged 6 years and older at baseline harboring disease-causing variants in the ABCA4 gene and with specified ocular lesions were enrolled from 9 centers in the United States and Europe. METHODS In the retrospective study, patients contributed medical record data from at least 2 and up to 4 visits for at least 1 examination modality: fundus autofluorescence (FAF), spectral-domain (SD) optical coherence tomography (SD OCT), and/or microperimetry (MP). The total observational period was at least 2 years and up to 5 years between single visits. Demographic and visual acuity (VA) data also were obtained. In the prospective study, eligible patients were examined at baseline using a standard protocol, with 6-month follow-up visits planned for a 2-year period for serial Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected VA, SD OCT, FAF, and MP. MAIN OUTCOME MEASURES Design and rationale of a multicenter study to determine the progression of STGD1 in 2 large retrospective and prospective international cohorts. Detailed baseline characteristics of both cohorts are presented, including demographics, and structural and functional retinal metrics. RESULTS Into the retrospective study, 251 patients (458 eyes) were enrolled; mean follow-up ± standard deviation was 3.9±1.6 years. At baseline, 36% had no or mild VA loss, and 47% of the study eyes had areas of definitely decreased autofluorescence (DDAF) with an average lesion area of 2.5±2.9 mm(2) (range, 0.02-16.03 mm(2)). Two hundred fifty-nine patients (489 eyes) were enrolled in the prospective study. At baseline, 20% had no or mild VA loss, and 64% had areas of DDAF with an average lesion area of 4.0±4.4 mm(2) (range, 0.03-24.24 mm(2)). The mean retinal sensitivity with MP was 10.8±5.0 dB. CONCLUSIONS The ProgStar cohorts have baseline characteristics that encompass a wide range of disease severity and are expected to provide valuable data on progression based on serial quantitative measurements derived from multiple methods, which will be critical to the design of planned clinical trials.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Biallelic Mutations in the Autophagy Regulator DRAM2 Cause Retinal Dystrophy with Early Macular Involvement

Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs(∗)3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165(∗)] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function.

Lamination of the Outer Plexiform Layer in Optic Atrophy Caused by Dominant WFS1 Mutations

Wolfram syndrome, or diabetes insipidus, diabetes mellitus, optic atrophy [OA], and deafness (DIDMOAD), is a neurodegenerative disorder with heterogeneous clinical manifestations caused by homozygous or compound heterozygous recessive mutations in the WFS1 gene (OMIM 606201). More recently, the phenotypic spectrum has expanded to include patients with dominant inheritance and limited clinical features, in particular OA in association with diabetes mellitus and/or sensorineural deafness. WFS1 encodes for an endoplasmic reticulum transmembrane protein, Wolframin, which is highly expressed in retinal tissues, including retinal ganglion cells, the photoreceptor inner segments, and the inner nuclear layer (INL) of the human eye, and mouse Müller cells. Thinning of the peripapillary nerve fiber and macular retinal ganglion cell layers are typical features of WFS1-related optic atrophy, but careful genotypeephenotype correlations have not yet been established. Herein, we report on a comprehensive macular optical coherence tomography (OCT) imaging study of 14 patients with OA secondary to mutations in WFS1 and the identification of a distinct outer plexiform layer (OPL) abnormality, which associates exclusively with dominant missense WFS1 mutations (Table 1, available at www.aaojournal.org). Averaged B-scans of 26 eyes from 14 patients with confirmed pathogenic WFS1 mutations were retrospectively reviewed from our Spectralis (Heidelberg Engineering Ltd., Heidelberg, Germany) databases. Our study had ethical and institutional approval and its design complied with the Declaration of Helsinki. A prominent feature in the macular spectral domain OCT images of 15 eyes from 8 patients harboring dominant WFS1 mutations (patients 1-8) was an abnormal reflectivity of the OPL, which was not observed in 11 eyes from 6 patients with recessive WFS1 mutations (Fig 1; Table 1, available at www.aaojournal.org). The OPL was composed of 3 distinct laminae: an innermost highly reflective lamina, a middle nonreflective lamina, and an outermost highly reflective lamina. To a certain extent, OPL thickness varied depending on the angle between the OCT laser beam and the retinal layers. Beam positions that were more perpendicular to the Henle’s fiber tracks resulted in a thicker OPL compared with beam positions that were parallel to these tracks, as illustrated by the left eye of patient 5 (Fig 1). However, in several eyes, an abnormally thick OPL was visualized, even with straight OCT beam positions, as for patient 6 (Fig 1). In the majority of patients, the middle OPL lamina formed a nearly confluent ring centered at the fovea and extending 1.0 to 1.5 mm from the foveolar center up to the optic disc. Perifoveal volumetric retinal scans were obtained for 22 eyes from 12 patients in our WFS1 cohort. Automated segmentation and retinal layer thickness analysis were carried out using the automated retinal Heidelberg Engineering segmentation tool included in the Spectralis Glaucoma Module software (version 6.0) after manual confirmation of each layer (Table 2, available at www.aaojournal.org). Marked thinning of the GCL-IPL complex was observed consistent with OA. All 11 eyes with abnormal OPL lamination had significantly thicker OPL and, more important, combined OPL and outer nuclear layer complex compared with control eyes, or the eyes of patients without OPL lamination. Compared with control eyes, INL thickness was increased in 4 eyes (Patients 2, 5, 8) with INL cystoid changes (Fig 1), but also in 5 other eyes with OPL lamination and in 3 eyes without OPL abnormality (Table 2, available at www.aaojournal.org). We have identified a previously unreported macular OCT feature in patients with Wolfram-related optic atrophy that is associated with dominant, but not recessive, WFS1 mutations. The lamination of the OPL was characterized by 3 distinct sublayers: (1) an innermost highly-reflective lamina, similar to what is frequently identified as the OPL in normal eyes; (2) a nonreflective cleft-like middle lamina; and (3) an outermost highlyreflective lamina with characteristics of Henle’s fiber layer. Our retrospective study did not enable a systematic evaluation of the effect of angle changes on the OPL reflectivity. Further work is needed to clarify whether the outermost lamina has truly abnormal reflectivity and whether the middle lamina represents an additional spatial structure or merely abnormal OCT reflectivity. However, as the thickness of combined OPL and outer nuclear layer complex should not be sensitive to OCT angle changes, its significantly increased thickness in eyes with abnormal OPL lamination suggests that an additional space could indeed account for the laminated OPL structure observed in this patient subgroup with dominant WFS1 mutations. Taking into consideration the spatial relationships between the 3 OPL laminae and previously published histologic studies, we propose that the middle cleft-like lamina is located between the synaptic and pedicles sublayers of the OPL. This region has also been shown to possess a particular retinal architecture with bending Müller cells. Wolframin is highly expressed in Müller cells and this cell type has been implicated in the development of OPL and INL edema, and changes within Henle’s fiber layer. The Müller cell is, therefore, an attractive candidate for the development of the observed OPL lamination. Interestingly, OPL lamination was associated only with dominant missense WFS1 mutations. It was not identified in any of the 6 patients harboring recessive homozygous or compound heterozygous WFS1 mutations. This distinct OPL abnormality has not been reported previously in the context of optic atrophy, and it could represent a specific deleterious effect of dominantWFS1 mutations. The missense variants identified in our patients are predicted to result in only a minor reduction in the wolframin protein level. Depletion of the native wild-type wolframin protein, which is thought to be the major pathophysiologic mechanism in recessive Wolfram syndrome, is therefore unlikely to be implicated in the etiology of the OPL lamination. Instead, dominant missense WFS1 mutations could result in a dysfunctional aberrant wolframin protein.

Novel heterozygous mutation in YAP1 in a family with isolated ocular colobomas

Congenital ocular colobomas are tissue defects which occur as a result of failure of the ectodermal optic vesicle fissure to close at around 5–7 weeks of fetal life.1 These defects can affect one o...

A novel missense mutation in both OPN1LW and OPN1MW cone opsin genes causes X-linked cone dystrophy (XLCOD5).

X-linked cone and cone-rod dystrophies (XLCOD and XLCORD) are an inherited group of retinal disorders primarily involving cone photoreceptors. The most common cause is mutation of RPGR. In a British family with XLCOD, we mapped the disorder to Xq26.1-qter, excluding RPGR and other known retinal degeneration genes. The cone opsin gene array on Xq28 was a positional candidate locus. A novel missense mutation (c.529T > C; p.W177R) was identified in exon 3 of both the long wavelength-sensitive (OPN1LW; LW, red) and medium wavelength-sensitive (OPN1MW; MW, green) cone opsin genes, which segregated with disease. Exon 3 sequences of both genes were identical, derived from the OPN1MW gene by partial gene conversion. The amino acid W177 is conserved in all opsins across species. We have shown that W177R in MW opsin results in protein misfolding and retention in the endoplasmic reticulum (ER). Mutations in the OPN1LW /OPN1MW cone opsin gene array can therefore cause a spectrum of phenotypes, from colour blindness to progressive cone dystrophy (XLCOD5).

Missense variants in the X-linked gene PRPS1 cause retinal degeneration in females.

Retinal dystrophies are a heterogeneous group of disorders of visual function leading to partial or complete blindness. We report the genetic basis of an unusual retinal dystrophy in five families with affected females and no affected males. Heterozygous missense variants were identified in the X‐linked phosphoribosyl pyrophosphate synthetase 1 (PRPS1) gene: c.47C > T, p.(Ser16Phe); c.586C > T, p.(Arg196Trp); c.641G > C, p.(Arg214Pro); and c.640C > T, p.(Arg214Trp). Missense variants in PRPS1 are usually associated with disease in male patients, including Arts syndrome, Charcot–Marie–Tooth, and nonsyndromic sensorineural deafness. In our study families, affected females manifested a retinal dystrophy with interocular asymmetry. Three unrelated females from these families had hearing loss leading to a diagnosis of Usher syndrome. Other neurological manifestations were also observed in three individuals. Our data highlight the unexpected X‐linked inheritance of retinal degeneration in females caused by variants in PRPS1 and suggest that tissue‐specific skewed X‐inactivation or variable levels of pyrophosphate synthetase‐1 deficiency are the underlying mechanism(s). We speculate that the absence of affected males in the study families suggests that some variants may be male embryonic lethal when inherited in the hemizygous state. The unbiased nature of next‐generation sequencing enables all possible modes of inheritance to be considered for association of gene variants with novel phenotypic presentation.

Retinal Dystrophy Gene Atlas

Describes a gene and all its possible clinical phenotypes and patient characteristics, along with retinal photos depicting each possible phenotype Written by prominent retinal dystrophy specialists from the largest dystrophy centers worldwide Contains more than 80 chapters, each of which describes the clinical and photographic manifestations of a specific gene Includes stunning clinical color photographs of the retina, autofluorescence imaging, and electrophysiologic findings and cross-sectional imaging Serves as a resource to aid genetic diagnosis in patients with retinal dystrophies by retina specialists and pediatric ophthalmologists in the United States, as well as hundreds of fellows and residents that

Jalili Syndrome: Cross-sectional and Longitudinal Features of Seven Patients With Cone-Rod Dystrophy and Amelogenesis Imperfecta

Purpose To characterize a series of 7 patients with cone-rod dystrophy (CORD) and amelogenesis imperfecta (AI) owing to confirmed mutations in CNNM4, first described as “Jalili Syndrome.” Design Retrospective observational case series. Methods Seven patients from 6 families with Jalili Syndrome were identified at 3 tertiary referral centers. We systematically reviewed their available medical records, spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence imaging (FAF), color fundus photography, and electrophysiological assessments. Results The mean age at presentation was 6.7 years (range 3-16 years), with 6 male and 1 female patient. CNNM4 mutations were identified in all patients. The mean Snellen best-corrected visual acuity (BCVA) at presentation was 20/246 (range 20/98 to 20/399) in the right eye and 20/252 (range 20/98 to 20/480) in the left. Nystagmus was observed in all 7 patients, and photophobia was present in 6. Funduscopic findings at presentation were variable, ranging from only mild disc pallor to retinal vascular attenuation and macular atrophy. Multimodal imaging demonstrated disease progression in all 7 patients over time. Electroretinography uniformly revealed progressive cone-rod dysfunction. Conclusions Jalili Syndrome is a rare CORD associated with AI. We have further characterized its ocular phenotype, including describing SD-OCT, FAF, and electrophysiological features; and report several novel disease-causing sequence variants. Moreover, this study presents novel longitudinal data demonstrating structural and functional progression over time, allowing better informed advice on prognosis.

The integrity and organization of the human AIPL1 functional domains is critical for its role as a HSP90-dependent co-chaperone for rod PDE6

Abstract Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) are associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy in early childhood. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. In this study, we characterized the functional deficits of AIPL1 variations, some of which induce aberrant pre-mRNA AIPL1 splicing leading to the production of alternative AIPL1 isoforms. We investigated the ability of the AIPL1 variants to mediate an interaction with HSP90 and modulate the rod cGMP PDE6 stability and activity. Our data revealed that both the FK506 binding protein (FKBP)-like domain and the tetratricopeptide repeat (TPR) domain of AIPL1 are required for interaction with HSP90. We further demonstrate that AIPL1 significantly modulates the catalytic activity of heterologously expressed rod PDE6. Although the N-terminal FKBP-like domain of AIPL1 binds the farnesylated PDE6α subunit through direct interaction with the farnesyl moiety, mutations compromising the integrity of the C-terminal TPR domain of AIPL1 also failed to modulate PDE6 activity efficiently. These AIPL1 variants moreover failed to promote the HSP90-dependent stabilization of the PDE6α subunit in the cytosol. In summary, we have successfully validated the disease-causing status of the AIPL1 variations in vitro. Our findings provide insight into the mechanism underlying the co-chaperone role of AIPL1 and will be critical for ensuring an early and effective diagnosis of AIPL1 LCA patients.