Insights Into PROM1-Macular Disease Using Multimodal Imaging.

Purpose: To describe the features of genetically confirmed PROM1-macular dystrophy in multimodal images. Methods: Thirty-six (36) eyes of 18 patients (5-66 years; mean age, 42.4 years) were prospectively studied by clinical examination and multimodal imaging. Short-wavelength autofluorescence (SW-AF) and quantitative fundus autofluorescence (qAF) images were acquired with a scanning laser ophthalmoscope (HRA+OCT, Heidelberg Engineering) modified by insertion of an internal autofluorescent reference. Further clinical testing included near-infrared autofluorescence (NIR-AF; HRA2, Heidelberg Engineering) with semiquantitative analysis, spectral domain-optical coherence tomography (HRA+OCT) and full-field electroretinography. All patients were genetically confirmed by exome sequencing. Results: All 18 patients presented with varying degrees of maculopathy. One family with individuals affected across two generations exhibited granular fleck-like deposits across the posterior pole. Areas of granular deposition in SW-AF and NIR-AF corresponded to intermittent loss of the ellipsoid zone, whereas discrete regions of hypoautofluorescence corresponded with a loss of outer retinal layers in spectral-domain optical coherence tomography scans. For 18 of the 20 eyes, qAF levels within the macula were within the 95% confidence intervals of healthy age-matched individuals; nor was the mean NIR-AF signal increased relative to healthy eyes. Conclusions: Although PROM1-macular dystrophy (Stargardt disease 4) can exhibit phenotypic overlap with recessive Stargardt disease, significantly increased SW-AF levels were not detected. As such, elevated bisretinoid lipofuscin may not be a feature of the pathophysiology of PROM1 disease. The qAF approach could serve as a method of early differential diagnosis and may help to identify appropriate disease targets as therapeutics become available to treat inherited retinal disease.

Phenotypic Variability of Retinal Disease Among a Cohort of Patients With Variants in the CLN Genes.

Purpose: To describe the phenotype of CLN-associated retinal dystrophy in a subset of patients at the Columbia University Medical Center, United States, and the Hospital das Clínicas de Pernambuco, Brazil, in comparison to the published literature. Methods: Eleven patients with confirmed biallelic variants in the CLN genes were evaluated via dilated fundus examination, clinical imaging, and full-field electroretinogram. A thorough literature search was conducted to determine previously published variants and associated phenotypes. Results: Genetic testing confirmed the presence of variants in CLN3, CLN7/MFSD8, CLN8, and GRN/CLN11. Five novel variants were identified, and four novel phenotypes of previously published alleles were described. The phenotype differed among patients with variants in the same gene and sometimes among patients with the same allele. Conclusions: Substantial phenotypic variability among variants in the CLN genes makes identification of genotype-phenotype or allele-phenotype correlations challenging. Further study is required to establish an extensive database for adequate patient counseling.

Chorioretinal atrophy following voretigene neparvovec despite the presence of fundus autofluorescence.

INTRODUCTION: Leber congenital amaurosis (LCA) type 2, due to disease-causing variants in RPE65, is characterized by severe visual loss in early infancy. Current treatments include voretigene neparvovec-rzyl (VN) for RPE65-associated LCA. Herein, we present the long-term follow-up of a patient treated with VN using quantitative autofluorescence (488 nm excitation). CASE REPORT: A 9-year-old girl with a diagnosis of LCA with biallelic variants in RPE65 presented for evaluation. The patient underwent VN treatment at the age of 11. The patient returned to clinic at age of 19 at which time imaging revealed evidence of chorioretinal atrophy. Quantitative autofluorescence performed prior to gene therapy and at 6- and 8-year follow-up revealed a central area of fundus autofluorescence. DISCUSSION: This case report demonstrates acquisition of fundus autofluorescence at 6- and 8-year follow-up despite the development of chorioretinal atrophy.

Expanding the phenotype of TTLL5-associated retinal dystrophy: a case series.

BACKGROUND: Inherited retinal dystrophies describe a heterogeneous group of retinal diseases that lead to the irreversible degeneration of rod and cone photoreceptors and eventual blindness. Recessive loss-of-function mutations in Tubulin Tyrosine Ligase Like 5 (TTLL5) represent a recently described cause of inherited cone-rod and cone dystrophy. This study describes the unusual phenotypes of three patients with autosomal recessive mutations in TTLL5. Examination of these patients included funduscopic evaluation, spectral-domain optical coherence tomography, short-wavelength autofluorescence, and full-field electroretinography (ffERG). Genetic diagnoses were confirmed using whole exome capture. Protein modeling of the identified variants was performed to explore potential genotype-phenotype correlations. RESULTS: Genetic testing revealed five novel variants in TTLL5 in three unrelated patients with retinal dystrophy. Clinical imaging demonstrated features of sectoral cone-rod dystrophy and cone dystrophy, with phenotypic variability seen across all three patients. One patient also developed high-frequency hearing loss during a similar time period as the onset of retinal disease, potentially suggestive of a syndromic disorder. Retinal structure findings were corroborated with functional measures including ffERG findings that supported these diagnoses. Modeling of the five variants suggest that they cause different effects on protein function, providing a potential reason for genotype-phenotype correlation in these patients. CONCLUSIONS: The authors report retinal phenotypic findings in three unrelated patients with novel mutations causing autosomal recessive TTLL5-mediated retinal dystrophy. These findings broaden the understanding of the phenotypes associated with TTLL5-mediated retinal disease and suggest that mutations in TTLL5 should be considered as a potential cause of sectoral retinal dystrophy in addition to cone-rod and cone dystrophies.

Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Allgrove syndrome with amyotrophy.

Allgrove syndrome is an autosomal recessive disease mostly caused by mutations in the AAAS gene. It has variable clinical features but its cardinal features comprise the triad of achalasia, alacrimia and adrenal insufficiency. It typically develops during the first decade of life, but some cases have second and third decades onset. We describe a 25-year-old woman with Allgrove syndrome who had progressive amyotrophy, achalasia, dry eyes and adrenal insufficiency since childhood. Awareness of its neurological manifestations and multisystem features helps to shorten the time for diagnosis and allow appropriate symptomatic treatment.

VITAMIN A DEFICIENCY MONITORED BY QUANTITATIVE SHORT WAVELENGTH FUNDUS AUTOFLUORESCENCE IN A CASE OF BARIATRIC SURGERY.

BACKGROUND/PURPOSE: Bariatric surgery is recognized as a treatment option for obesity. However, the cost-efficiency of screening for serum vitamin A and the effectiveness of its oral supplementation in these patients remain unclear. Here, we report a case in which vitamin A and carotenoid deficiency after bariatric surgery were monitored by noninvasive quantitative fundus autofluorescence imaging. METHODS: Case report. RESULTS: A 62-year-old man presented with a history of progressive night blindness. He had duodenal switch surgery 13 years earlier. One year before the initial visit, he had begun oral supplements of vitamins A. Short wavelength fundus autofluorescence images acquired for quantitative fundus autofluorescence revealed an intensity that was lower than the healthy-eye range. Scotopic rod-specific full-field electroretinograms were extinguished. These findings were consistent with vitamin A deficiency. The patient was given intramuscular vitamin A injections. At follow-up, quantitative fundus autofluorescence improved, ERG increased to normal, but macular pigment was unchanged. CONCLUSION: Oral vitamin A supplementation may not be sufficient after mal-absorptive surgery and a quantitative and noninvasive short wavelength fundus autofluorescence imaging technique may be useful to monitor the status of vitamin A and the carotenoids comprising macular pigment in the retina.

A mutation in CRX causing pigmented paravenous retinochoroidal atrophy.

INTRODUCTION: Mutations in the cone-rod homeobox (CRX) gene, a known cause of inherited retinal dystrophy, are characterized by extensive phenotypic heterogeneity. We describe a novel presentation of rod-cone dystrophy (RCD) phenocopying pigmented paravenous retinochoroidal atrophy associated with a mutation in CRX. CASE DESCRIPTION: A 53-year-old man and his 48-year-old brother presented with a history of progressive vision loss and nyctalopia. Fundus examination revealed a bull's eye lesion with chorioretinal atrophy and intraretinal pigment migration, while spectral-domain optical coherence tomography (SD-OCT) demonstrated retinal thinning with outer retinal atrophy. On short-wavelength autofluorescence (SW-AF) imaging, an atypical paravenous pattern of atrophy with a surrounding hyperautofluorescent border was observed. Full-field electroretinogram (ffERG) revealed a rod-cone pattern of dysfunction. A heterozygous pathogenic variant, c.119G>A:p.(Arg40Gln), in the CRX gene was identified in both brothers and segregated in their family. CONCLUSION: This case report broadens the currently known phenotypic presentations of CRX-associated retinopathy and suggests that mutations in CRX may be associated with pigmented paravenous retinochoroidal atrophy.

Retinal Manifestations of Mitochondrial Oxidative Phosphorylation Disorders.

Purpose: The purpose of this paper was to discuss manifestations of primary mitochondrial dysfunctions and whether the retinal pigment epithelium or the photoreceptors are preferentially affected. Methods: A retrospective analysis was performed of patients with clinically and laboratory confirmed diagnoses of maternally inherited diabetes and deafness (MIDD) or Kearns-Sayre syndrome (KSS). Patients underwent full ophthalmic examination, full-field electroretinogram, and multimodal imaging studies, including short-wavelength autofluorescence, spectral domain-optical coherence tomography, and color fundus photography. Results: A total of five patients with MIDD and four patients with KSS were evaluated at two tertiary referral centers. Mean age at initial evaluation was 50.3 years old. Nascent outer retinal tubulations corresponding with faint foci of autofluorescence were observed in two patients with MIDD. Characteristic features of this cohort included a foveal sparing phenotype observed in 13 of 18 eyes (72%), global absence of intraretinal pigment migration, and preserved retinal function on full-field electroretinogram testing in 12 of 16 eyes (75%). One patient diagnosed with MIDD presented with an unusual pattern of atrophy surrounding the parapapillary region and one patient with KSS presented with an atypical choroideremia-like phenotype. Conclusions: MIDD and KSS are phenotypically heterogeneous disorders. Several features of disease suggest that primary mitochondrial dysfunction may first affect the retinal pigment epithelium followed by secondary photoreceptor loss. Similarities between primary mitochondrial degenerations and retinal disorders, such as age-related macular degeneration may suggest a primary role of mitochondria in the pathogenesis of these oligogenic disorders.

Quantitative Fundus Autofluorescence in HCQ Retinopathy.

Purpose: To increase our understanding of the mechanisms underlying hydroxychloroquine (HCQ) retinopathy, analyses by quantitative fundus autofluorescence (qAF) and near-infrared fundus autofluorescence (NIR-AF) were compared to results obtained by recommended screening tests. Methods: Thirty-one patients (28 females, 3 males) were evaluated with standard automated perimetry and spectral domain optical coherence tomography (SD-OCT); 28 also had multifocal electroretinography (mfERG). Measurement of short-wavelength fundus autofluorescence (SW-AF) by qAF involved the use of an internal fluorescent reference and intensity measurements in eight concentric segments at 7° to 9° eccentricity. For semiquantitative analysis of NIR-AF, intensities were acquired along a vertical axis through the fovea. Results: Four of 15 high-dose (total dose >1000 g, daily dose >5.0 mg/kg) patients and one of 16 low-dose (total dose <1000 g, daily dose 4.4 mg/kg) patients were diagnosed with HCQ-associated retinopathy based on abnormal 10-2 visual fields, SD-OCT, and SW-AF imaging. Three of the high-dose patients also had abnormal mfERG results. Of the five patients exhibiting retinopathy, two had qAF color-coded images revealing higher intensities inferior, nasal, and lateral to the fovea. The abnormal visual fields also exhibited superior-inferior differences. Mean NIR-AF gray-level intensities were increased in four high-dose patients with no evidence of retinopathy. In two patients with retinopathy, NIR-AF intensity within the parafovea was below the normal range. One high-dose patient (6.25 mg/kg) had only abnormal mfERG results. Conclusions: These findings indicate that screening for HCQ retinopathy should take into consideration superior-inferior differences in susceptibility to HCQ retinopathy.

Optical coherence tomography in the evaluation of retinitis pigmentosa.

BACKGROUND: Optical coherence tomography (OCT) is a non-invasive imaging test that provides easily obtainable and highly reproducible cross-sectional images of the retina. Improved modalities of the OCT that are capable of providing high quality images of not only the retina, but also the deeper structures and vasculature have been developed, including swept-source OCTs and OCT angiography. MATERIALS AND METHODS: Review. RESULTS: The use of OCT in the monitoring of retinitis pigmentosa has been well described and numerous signs of disease progression have been studied. Notably among them are the detection of changes to retinal thickness, the ellipsoid zone, the vasculature on OCT angiography, and cystoid macular edema. CONCLUSION: In this review, we discuss the multiple applications of OCT as a tool in the monitoring of retinitis pigmentosa and its potential use as an outcome measurement in current and future therapeutic endeavors.

Short-Wavelength and Near-Infrared Autofluorescence in Patients with Deficiencies of the Visual Cycle and Phototransduction.

Fundus autofluorescence is a valuable imaging tool in the diagnosis of inherited retinal dystrophies. With the advent of gene therapy and the numerous ongoing clinical trials for inherited retinal degenerations, quantifiable and reliable outcome measurements continually need to be identified. In this retrospective analysis, normalized and non-normalized short-wavelength (SW-AF) and near-infrared (NIR-AF) autofluorescence images of ten patients with mutations in visual cycle (VC) genes and nineteen patients with mutations in phototransduction (PT) genes were analyzed. Normalized SW-AF and NIR-AF images appeared darker in all patients with mutations in the VC as compared to patients with mutations in PT despite the use of significantly higher detector settings for image acquisition in the former group. These findings were corroborated by quantitative analysis of non-normalized SW-AF and NIR-AF images; signal intensities were significantly lower in all patients with mutations in VC genes as compared to those with mutations in PT genes. We conclude that qualitative and quantitative SW-AF and NIR-AF images can serve as biomarkers of deficiencies specific to the VC. Additionally, quantitative autofluorescence may have potential for use as an outcome measurement to detect VC activity in conjunction with future therapies for patients with mutations in the VC.

Differences in Intraretinal Pigment Migration Across Inherited Retinal Dystrophies.

PURPOSE: To determine whether there are differences in the prevalence of intraretinal pigment migration (IPM) across ages and genetic causes of inherited retinal dystrophies (IRDs). DESIGN: Retrospective cohort study. METHODS: Patients were evaluated at a single tertiary referral center. All patients with a clinical diagnosis of IRD and confirmatory genetic testing were included in these analyses. A total of 392 patients fit inclusion criteria, and 151 patients were excluded based on inconclusive genetic testing. Patients were placed into 3 groups, ciliary and ciliary-related photoreceptor, nonciliary photoreceptor, and retinal pigment epithelium (RPE), based on the cellular expression of the gene and the primary affected cell type. The presence of IPM was evaluated by using slit lamp biomicroscopy, indirect ophthalmoscopy, and wide-field color fundus photography. RESULTS: IPM was seen in 257 of 339 patients (75.8%) with mutations in photoreceptor-specific genes and in 18 of 53 patients (34.0%) with mutations in RPE-specific genes (P < .0001). Pairwise analysis following stratification by age and gene category suggested significant differences at all age groups between patients with mutations in photoreceptor-specific genes and patients with mutations in RPE-specific genes (P < .05). A fitted multivariate logistic regression model was produced and demonstrated that the incidence of IPM increases as a function of both age and gene category. CONCLUSIONS: IPM is a finding more commonly observed in IRDs caused by mutations in photoreceptor-specific genes than RPE-specific genes. The absence of IPM does not always rule out IRD and should raise suspicion for disease mutations in RPE-specific genes.

Optical Gap Biomarker in Cone-Dominant Retinal Dystrophy.

PURPOSE: To characterize the progression of optical gaps and expand the known etiologies of this phenotype. DESIGN: Retrospective cohort study. METHODS: Thirty-six patients were selected based on the identification of an optical gap on spectral-domain optical coherence tomography (OCT) from a large cohort of patients (N = 746) with confirmed diagnoses of inherited retinal dystrophy. The width and height of the gaps in 70 eyes of 36 patients were measured by 2 independent graders using the caliper tool on Heidelberg Explorer. Measurements of outer and central retinal thickness were also evaluated and correlated with gap dimensions. RESULTS: Longitudinal analysis confirmed the progressive nature of optical gaps in patients with Stargardt disease, achromatopsia, occult macular dystrophy, and cone dystrophies (P < .003). Larger changes in gap width were noted in patients with Stargardt disease (78.1 µm/year) and cone dystrophies (31.9 µm/year) compared with patients with achromatopsia (16.2 µm/year) and occult macular dystrophy (15.4 µm/year). Gap height decreased in patients with Stargardt disease (6.5 µm/year; P = .02) but increased in patients with achromatopsia (3.3 µm/year) and occult macular dystrophy (1.2 µm/year). Gap height correlated with measurements of central retinal thickness at the fovea (r = 0.782, P = .00012). Interocular discordance of the gap was observed in 7 patients. Finally, a review of all currently described etiologies of optical gap was summarized. CONCLUSION: The optical gap is a progressive phenotype seen in an increasing number of etiologies. This progressive nature suggests a use as a biomarker in the understanding of disease progression. Interocular discordance of the phenotype may be a feature of Stargardt disease and cone dystrophies.

Comparative Analysis of Functional and Structural Decline in Retinitis Pigmentosas.

Retinitis pigmentosa (RP) is a category of inherited retinal dystrophies that is best prognosticated using electroretinography (ERG). In this retrospective cohort study of 25 patients with RP, we evaluated the correlation between 30 Hz flicker ERG and structural parameters in the retina. Internationally standardized 30 Hz flicker ERG recordings, short-wavelength autofluorescence (SW-AF), and spectral domain-optical coherence tomography (SD-OCT) were acquired at two visits at least one year apart. Vertical and horizontal hyperautofluorescent ring diameter measurements with SW-AF, as well as ellipsoid zone (EZ) line width measurements with SD-OCT, were used as structural parameters of disease progression. The 30 Hz flicker ERG amplitude decreased by 2.2 ± 0.8 µV/year (p = 0.011), while implicit times remained unchanged. For SD-OCT, the EZ line decreased by 204.1 ± 34.7 µm/year (p < 0.001). Horizontal and vertical hyperautofluorescent ring diameters decreased by 161.9 ± 25.6 µm/year and 146.9 ± 34.6 µm/year, respectively (p = 0.001), with SW-AF. A correlation was found between the progression rates of the 30 Hz flicker amplitude recorded with Burian-Allen electrodes and both the horizontal ring diameter (p = 0.020) and EZ line (p = 0.044). SW-AF and SD-OCT, two readily available imaging techniques, may be used to prognosticate disease progression because of the reliability of their measurements and correlation with functional outcome.

PKM2 ablation enhanced retinal function and survival in a preclinical model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a neurodegenerative disorder that causes irreversible vision loss in over 1.5 million individuals world-wide. The genetic heterogeneity of RP necessitates a broad therapy that is able to provide treatment in a gene- and mutation- non-specific manner. In this study, we identify the therapeutic benefits of metabolic reprogramming by targeting pyruvate kinase M2 (PKM2) in a Pde6ß preclinical model of RP. The genetic contributions of PKM2 inhibition in retinal degeneration were evaluated through histology and electroretinogram (ERG) followed by a statistical analysis using a linear regression model. Notably, PKM2 ablation resulted in thicker retinal layers in Pde6ß-mutated mice as compared to the controls, suggesting greater photoreceptor survival. Consistent with these anatomical findings, ERG analyses revealed that the maximum b-wave is on average greater in Pkm2 knockout mice than in mice with intact Pkm2, indicating enhanced photoreceptor function. These rescue phenotypes from Pkm2 ablation in a preclinical model of RP indicate that a metabolome reprogramming may be useful in treating RP.

Sequential multiple retinal vein occlusions and transient ischemic attack in MTHFR polymorphism and protein S deficiency.

BACKGROUND: The C677T variant of the MTHFR (5,10-Methylenetetrahydrofolate reductase) gene is associated with increased susceptibility to homocystinuria (OMIM#236250), neural tube defects (OMIM#601634), schizophrenia (OMIM#181500), thromboembolism (OMIM#188050), and vascular diseases. Protein S deficiency is also associated with an increased risk of thromboembolism from reduced thrombin generation. In this report, we describe the case of a patient who presented with multiple retinal vein occlusions likely caused by an underlying combination of a homozygous MTHFR C677T variant and protein S deficiency. METHODS: We performed 8 years of continuous ophthalmic follow-up of one patient diagnosed with central retinal vein occlusion. Peripheral blood was collected for metabolic evaluation and hypercoagulability assessment. Targeted gene sequencing was used for genetic diagnosis. Examination of the retinal vasculature was performed through dilated funduscopic examination, digital color fundus and ultrawide-field color fundus photography, spectral domain optical coherence tomography, and fluorescein angiography. RESULTS: Sequential retinal vein occlusions and a transient ischemic attack were observed during the follow-up period. Targeted gene sequencing by PCR identified the homozygous MTHFR C677T variant. The metabolic profile indicated low-protein S activity, high levels of vitamin B6, and LDL cholesterol consistent with her hypercoagulable state. Prescription of low-dose aspirin and atorvastatin for hypercholesterolemia resulted in no further neovascularization, leakage, or vein occlusion. CONCLUSION: Retinal vein occlusions associated with the MTHFR C677T variant and protein S deficiency may signal impending systemic thromboembolic episodes and warrant aggressive preventative measures.

Effects of deficiency in the RLBP1-encoded visual cycle protein CRALBP on visual dysfunction in humans and mice.

Mutations in retinaldehyde-binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that is compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp1/Cralbp-/- mice. In the RLBP1/CRALBP-affected individuals, nonrecordable rod-specific electroretinogram traces were recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain-optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands. In short-wavelength fundus autofluorescence (SW-AF) images, speckled hyperautofluorescence and mottling indicated macular involvement. In both the affected individuals and their asymptomatic carrier parents, reduced SW-AF intensities, measured as quantitative fundus autofluorescence (qAF), indicated chronic impairment in 11-cis-retinal availability and provided information on mutation severity. Hypertransmission of the SD-OCT signal into the choroid together with decreased near-infrared autofluorescence (NIR-AF) provided evidence for retinal pigment epithelial cell (RPE) involvement. In Rlbp1/Cralbp-/- mice, reduced 11-cis-retinal levels, qAF and NIR-AF intensities, and photoreceptor loss were consistent with the clinical presentation of the affected siblings. These findings indicate that RLBP1 mutations are associated with progressive disease involving RPE atrophy and photoreceptor cell degeneration. In asymptomatic carriers, qAF disclosed previously undetected visual cycle deficiency.

Fundoscopy-directed genetic testing to re-evaluate negative whole exome sequencing results.

BACKGROUND: Whole exome sequencing (WES) allows for an unbiased search of the genetic cause of a disease. Employing it as a first-tier genetic testing can be favored due to the associated lower incremental cost per diagnosis compared to when using it later in the diagnostic pathway. However, there are technical limitations of WES that can lead to inaccurate negative variant callings. Our study presents these limitations through a re-evaluation of negative WES results using subsequent tests primarily driven by fundoscopic findings. These tests included targeted gene testing, inherited retinal gene panels, whole genome sequencing (WGS), and array comparative genomic hybridization. RESULTS: Subsequent genetic testing guided by fundoscopy findings identified the following variant types causing retinitis pigmentosa that were not detected by WES: frameshift deletion and nonsense variants in the RPGR gene, 353-bp Alu repeat insertions in the MAK gene, and large exonic deletion variants in the EYS and PRPF31 genes. Deep intronic variants in the ABCA4 gene causing Stargardt disease and the GUCY2D gene causing Leber congenital amaurosis were also identified. CONCLUSIONS: Negative WES analyses inconsistent with the phenotype should raise clinical suspicion. Subsequent genetic testing may detect genetic variants missed by WES and can make patients eligible for gene replacement therapy and upcoming clinical trials. When phenotypic findings support a genetic etiology, negative WES results should be followed by targeted gene sequencing, array based approach or whole genome sequencing.