Atypical retinopathy in ataxia with vitamin E deficiency: report of a sibship.

Typical retinitis pigmentosa (RP) may not be the only retinal phenotype encountered in ataxia with vitamin E deficiency (AVED). The following short case series describes a novel form of retinopathy in AVED. We describe two patients with AVED belonging to the same consanguineous sibship. Both presented an unusual retinopathy consisting of scattered, multifocal, nummular, hyperautofluorescent atrophic retinal patches. The retinopathy remained stable under vitamin E supplementation. We hypothesize these changes to be the result of arrested AVED-related RP following early supplementation with α-tocopherol acetate.

Ocular manifestations of the genetic causes of focal and segmental glomerulosclerosis.

Genetic forms of focal and segmental glomerulosclerosis (FSGS) often have extra-renal manifestations. This study examined FSGS-associated genes from the Genomics England Renal proteinuria panel for reported and likely ocular features. Thirty-two of the 55 genes (58%) were associated with ocular abnormalities in human disease, and a further 12 (22%) were expressed in the retina or had an eye phenotype in mouse models. The commonest genes affected in congenital nephrotic syndrome (NPHS1, NPHS2, WT1, LAMB2, PAX2 but not PLCE1) may have ocular manifestations . Many genes affected in childhood-adolescent onset FSGS (NPHS1, NPHS2, WT1, LAMB2, SMARCAL1, NUP107 but not TRPC6 or PLCE1) have ocular features. The commonest genes affected in adult-onset FSGS (COL4A3-COL4A5, GLA ) have ocular abnormalities but not the other frequently affected genes (ACTN4, CD2AP, INF2, TRPC6). Common ocular associations of genetic FSGS include cataract, myopia, strabismus, ptosis and retinal atrophy. Mitochondrial forms of FSGS (MELAS, MIDD, Kearn's Sayre disease) are associated with retinal atrophy and inherited retinal degeneration. Some genetic kidney diseases (CAKUT, ciliopathies, tubulopathies) that result in secondary forms of FSGS also have ocular features. Ocular manifestations suggest a genetic basis for FSGS, often help identify the affected gene, and prompt genetic testing. In general, ocular abnormalities require early evaluation by an ophthalmologist, and sometimes, monitoring or treatment to improve vision or prevent visual loss from complications. In addition, the patient should be examined for other syndromic features and first degree family members assessed.

An incipient late-onset retinal degeneration with a C1QTNF5 mutation: a case report with an 11-year follow-up.

PURPOSE: The purpose of this study was to describe and diagnose the difficulty in a long-term follow-up (eleven years) patient with a very early presentation of late-onset retinal degeneration (L-ORD) and the significance of electrophysiological examinations and follow-up in assessing undiagnosed inherited retinal diseases. METHODS: This is an observational case report of a 56-year-old woman, with scattered multiple yellow-white retinal dots firstly diagnosed as fundus albipunctatus. Ten years after presentation, a deterioration in rod and cone responses in ff-ERG was detected, which allowed us to discard the first diagnostic hypothesis and proceed with a genetic testing. RESULTS: Ten years after presentation, she presented a clear progression of the abnormal photoreceptor response with a cone and rod involvement in ff-ERG, which was not compatible with the previous suspicion of fundus albipunctatus. Six months later, genetic testing results together with the typical progression of atrophic patchy lesions in multimodal imaging allowed a certain diagnosis of L-ORD, caused by an already reported pathogenic variant in the C1QTNF5 gene (c.563C > T; p. Pro188 Leu). CONCLUSIONS: We demonstrate the importance of the ff-ERG examination and the follow-up (or ERG and imaging repetition) in the differential diagnosis of an incipient L-ORD, which can be easily misdiagnosed in the early stages, before the appearance of the characteristic chorioretinal atrophy seen with the progression of this rare disease.

Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina.

Inherited retinal dystrophies are often associated with mutations in the genes involved in the phototransduction cascade in photoreceptors, a paradigmatic signaling pathway mediated by G protein-coupled receptors. Photoreceptor viability is strictly dependent on the levels of the second messengers cGMP and Ca2+. Here we explored the possibility of modulating the phototransduction cascade in mouse rods using direct or liposome-mediated administration of a recombinant protein crucial for regulating the interplay of the second messengers in photoreceptor outer segments. The effects of administration of the free and liposome-encapsulated human guanylate cyclase-activating protein 1 (GCAP1) were compared in biological systems of increasing complexity (in cyto, ex vivo, and in vivo). The analysis of protein biodistribution and the direct measurement of functional alteration in rod photoresponses show that the exogenous GCAP1 protein is fully incorporated into the mouse retina and photoreceptor outer segments. Furthermore, only in the presence of a point mutation associated with cone-rod dystrophy in humans p.(E111V), protein delivery induces a disease-like electrophysiological phenotype, consistent with constitutive activation of the retinal guanylate cyclase. Our study demonstrates that both direct and liposome-mediated protein delivery are powerful complementary tools for targeting signaling cascades in neuronal cells, which could be particularly important for the treatment of autosomal dominant genetic diseases.

Genetic Testing of Patients with Inherited Retinal Diseases in the European Countries: An International Survey by the European Vision Institute.

INTRODUCTION: The purpose of this project was to explore the current standards of clinical care genetic testing and counseling for patients with inherited retinal diseases (IRDs) from the perspective of leading experts in selected European countries. Also, to gather opinions on current bottlenecks and future solutions to improve patient care. METHODS: On the initiative of the European Vision Institute, a survey questionnaire with 41 questions was designed and sent to experts in the field from ten European countries. Each participant was asked to answer with reference to the situation in their own country. RESULTS: Sixteen questionnaires were collected by November 2023. IRD genetic tests are performed in clinical care settings for 80% or more of tested patients in 9 countries, and the costs of genetic tests in clinical care are covered by the public health service to the extent of 90% or more in 8 countries. The median proportion of patients who are genetically tested, the median rate of genetically solved patients among those who are tested, and the median proportion of patients receiving counseling are 51-70%, 61-80%, and 61-80%, respectively. Improving the education of healthcare professionals who facilitate patient referrals to specialized centers, improving access of patients to more thorough genotyping, and increasing the number of available counselors were the most advocated solutions. CONCLUSION: There is a significant proportion of IRD patients who are not genetically tested, whose genetic testing is inconclusive, or who do not receive counseling. Educational programs, greater availability of state-of-the-art genotyping and genetic counselors could improve healthcare for IRD patients.

Limited Added Diagnostic Value of Whole Genome Sequencing in Genetic Testing of Inherited Retinal Diseases in a Swiss Patient Cohort.

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields.

Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function.

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.

Full-field Scotopic Threshold Improvement after Voretigene Neparvovec-rzyl Treatment Correlates with Chorioretinal Atrophy.

PURPOSE: To analyze demographic and ophthalmic data in patients with and without chorioretinal atrophy after voretigene neparvovec-rzyl (VN) to identify possible causes for this phenomenon. DESIGN: Retrospective cohort study with longitudinal follow-up. PARTICIPANTS: A total of 71 eyes of 38 patients aged 2 to 44 years with RPE65-mediated retinal dystrophy treated with VN across 2 large gene therapy centers in the United States and Germany. METHODS: Patients treated with VN who developed atrophy were compared with those who did not. MAIN OUTCOME MEASURES: Gender, age, surgical center, spherical equivalent refraction, best-corrected visual acuity (BCVA), baseline full-field scotopic threshold testing (FST), and posttreatment change in FST. RESULTS: A total of 20 eyes of 12 patients developed atrophy after treatment with VN (28% of all eyes). There was no significant difference in gender, age, surgical center, or spherical equivalent refraction between the atrophy group and the no atrophy group. However, patients between school age and young adulthood were predominantly affected, whereas the youngest and the oldest patients did not develop atrophy. Baseline BCVA was better in patients who developed atrophy than those who did not (P = 0.006). The postoperative improvement in FST at 1 month was significantly higher in the atrophy group than in the no atrophy group (P = 0.0005), and this difference remained statistically significant at 1 year (P = 0.0001). There was no correlation to baseline FST, to inflammation, or to which eye was treated first. CONCLUSIONS: The degree of FST improvement after VN appears to be strongly correlated with the development of VN-related chorioretinal atrophy. This finding raises the possibility that atrophy may develop as a toxic or metabolic sequela of vector-mediated RPE65 expression. In light of the expanding number of retinal gene therapy clinical trials, this complication warrants further study because it may not be limited to VN. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Phenotypic variability in PRPH2 as demonstrated by a family with incomplete penetrance of autosomal dominant cone-rod dystrophy.

INTRODUCTION: Mutations in the peripherin-2 gene (PRPH2) are a common cause of inherited retinal dystrophies well known for their phenotypic diversity. We describe a novel presentation of the c.623G > A; p.(Gly208Asp) variant in association with cone-rod dystrophy and reduced penetrance. CASE DESCRIPTION: A 39-year-old man presents with a history of decreased visual acuity, photophobia, and dyschromatopsia. Fundus examination was largely unremarkable while spectral-domain optical coherence tomography (SD-OCT) demonstrated diffuse granularity at the ellipsoid zone. Full-field electroretinogram (ffERG) revealed a cone-rod dystrophy. Genetic testing revealed a heterozygous pathogenic variant, c.623G > A; p.(Gly208Asp), in the PRPH2 gene, also found in an unaffected brother. The 50-year-old brother had no visual symptoms and no findings on fundus examination. SD-OCT showed normal retinal architecture and ffERG was within normal limits bilaterally. CONCLUSION: This case report broadens the known phenotypic presentations of PRPH2-associated retinopathy and suggests that the PRPH2 variant c.623G > A; p.(Gly208Asp) may be associated with reduced penetrance.

Unraveling mucolipidosis type III gamma through whole genome sequencing in late-onset retinitis pigmentosa: a case report.

BACKGROUND: We describe the case of a 47-year-old man referred to a retinal clinic and diagnosed with late-onset retinitis pigmentosa. Surprisingly, genetic testing revealed compound heterozygous pathogenic variants in GNPTG, leading to the diagnosis of the autosomal recessive lysosomal storage disorder mucolipidosis type III gamma. Mucolipidosis type III gamma is typically diagnosed during childhood due to symptoms relating to skeletal dysplasia. Retinal dystrophy is not a common phenotypic feature. CASE PRESENTATION: Ophthalmologic examination was consistent with a mild form of retinitis pigmentosa and included fundus photography, measurement of best-corrected visual acuity, optical coherence tomography, electroretinogram and visual field testing. Extraocular findings included joint restriction and pains from an early age leading to bilateral hip replacement by age 30, aortic insufficiency, and hypertension. Genetic analysis was performed by whole genome sequencing filtered for a gene panel of 325 genes associated with retinal disease. Two compound heterozygous pathogenic variants were identified in GNPTG, c.347_349del and c.607dup. The diagnosis of mucolipidosis type III gamma was confirmed biochemically by measurement of increased activities of specific lysosomal enzymes in plasma. CONCLUSION: To our knowledge this is the first description of retinitis pigmentosa caused by compound heterozygous variants in GNPTG, providing further indications that late-onset retinal dystrophy is part of the phenotypic spectrum of mucolipidosis type III gamma.

The Role of Microglia in Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are a leading cause of irreversible visual loss in the developed world. The primary driver of pathology in IRDs is pathogenic genetic variant. However, there is increasing evidence, from recent studies, for a role of the immune system in disease mechanism, particularly retinal microglia. Microglia are the primary immune cells in the retina and actively contribute to disease pathogenesis when activated locally by phagocytosing photoreceptors, inducing inflammation and signaling infiltration of circulating monocytes. In this article, we discuss the evidence for the contribution of retinal microglia to IRD pathogenesis reported so far using mice model.

Genotypic and phenotypic characterisation of RP2- and RPGR-associated X-linked inherited retinal dystrophy, including female manifestations.

BACKGROUND: With the promise of gene replacement therapy, eligible males and females with X-linked inherited retinal dystrophy (XL-IRD) should be identified. METHODS: Retrospective observational cohort study to establish the phenotypic and genotypic spectrum of XL-IRD within New Zealand (NZ). Thirty-two probands, including 9 females, with molecularly proven XL-IRD due to RP2 or RPGR mutations, and 72 family members, of which 43 were affected, were identified from the NZ IRD Database. Comprehensive ophthalmic phenotyping, familial cosegregation, genotyping, and bioinformatics were undertaken. Main outcome measures were: RP2 and RPGR pathogenic variant spectrum, phenotype in males and females (symptoms, age of onset, visual acuity, refraction, electrophysiology, autofluorescence, retinal appearance), and genotype-phenotype correlation. RESULTS: For 32 families, 26 unique pathogenic variants were identified; in RP2 (n = 6, 21.9% of all families), RPGR exons 1-14 (n = 10, 43.75%), and RPGR-ORF15 (n = 10, 34.3%). Three RP2 and 8 RPGR exons 1-14 variants are novel, rare, and cosegregate. Thirty-one percent of carrier females were significantly affected, with 18.5% of families initially classified as autosomal dominant. Of five Polynesian families, 80% had novel disease-causing variants. One Maori family showed keratoconus segregating with an ORF15 variant. CONCLUSIONS: Significant disease was present in 31% of genetically proven female carriers, often leading to an erroneous presumption of the inheritance pattern. Pathogenic variants in 44% of the families were in exon 1-14 of RPGR, more frequent than usually described, which may inform the gene testing algorithm. Proving cosegregation in families for novel variants and identifying affected females and males translates to optimised clinical care and potential for gene therapy.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Inflammation after Voretigene Neparvovec Administration in Patients with RPE65-Related Retinal Dystrophy.

PURPOSE: To report on the prevalence of intraocular inflammation after subretinal voretigene neparvovec (VN) administration. DESIGN: Retrospective review of medical files. PARTICIPANTS: All patients receiving VN in Denmark. METHODS: Twelve patients received VN gene therapy as standard of care for biallelic RPE65-related retinal disease. Bilateral treatment was performed in 11 patients and unilateral treatment in 1 patient. Patients were followed clinically before and after VN administration using functional measurements (visual acuity, full-field scotopic threshold test, visual fields) and structural evaluations (fundus imaging [color and autofluorescence], OCT, slit-lamp). MAIN OUTCOME MEASURES: Signs of intraocular inflammation, including vitritis and outer retinal infiltrates. RESULTS: Vitritis was observed in 9 of 23 eyes receiving VN. The median time to resolution of vitritis from the time of treatment was 89 days. Four eyes also presented with outer retinal infiltrates at the time of vitritis. Inflammation subsided on immunosuppressant therapy. The presence of inflammation did not adversely affect visual outcome after VN therapy. In 1 eye, outer retinal infiltrates were demonstrated to precede later development of atrophy. CONCLUSIONS: Patients undergoing subretinal gene therapy need to be closely monitored for signs of inflammation. Although we did not observe a detrimental effect on visual function in eyes with inflammation, it seems wise to treat it appropriately because it may lead to atrophy of the retinal pigment epithelium and outer retina. Also, it seems advisable to reduce the inflammatory load, such as using a surgical technique that minimizes residual viral vectors in the vitreous body.

Optical coherence tomography angiography (OCT-A) in retinitis pigmentosa and macular dystrophy patients: a retrospective study.

PURPOSE: To evaluate macular vascular abnormalities in patients with macular dystrophies (MD) and retinitis pigmentosa (RP) through application of optical coherence tomography angiography (OCT-A). METHODS: In this retrospective study, patients with MD and RP were examined by OCT-A and compared to healthy individuals. OCT-A images were analyzed regarding the diameter and surface area of the foveal avascular zone (FAZ) as well as flow (FL) in different retinal layers (superficial vascular complex (SVC), intermediate capillary complex (ICP), deep capillary complex (DCP), choriocapillaris (CC), and choroid (CD)). RESULTS: Twenty-one patients with MD, 21 patients with RP without macular edema (RPnE), 8 patients with RP with edema (RPwE), and 41 healthy individuals were enrolled. The group of MD and RPnE patients showed none or only minor changes in FAZ. In RPwE patients, the FAZ was significantly smaller in vertical and horizontal measurements and surface area in SVC, whereas it was markedly enlarged in ICP. FL was significantly reduced compared to healthy individuals by an average of 13.2% in CD, 14.2% in CC, and 8.4% in DCP in all patient groups. In ICP, the reduction was 9.2% for RPnE and 12.7% for RPwE patients. The SVC showed reduced FL in the MD (8.1%) and RPnE (10.3%) group. CONCLUSIONS: OCT-A is a valuable tool to examine retinal vascular abnormalities in patients with MD and RP. OCT-A revealed a reduced flow in various retinal layers in MD, RPnE, and RPwE. Alterations of the FAZ were less distinct in these groups which add to the variation reported previously.

Longitudinal analysis of health care costs in patients with childhood onset inherited retinal dystrophies compared to healthy controls.

BACKGROUND: We evaluated health care costs in patients with childhood onset visual impairment caused by inherited retinal dystrophies (IRD). METHODS: The IRD cohort, identified from the Danish Registry of Blind and Partially Sighted Children, was compared to age- and sex-matched controls from the national, Danish population registry. Information on health care expenditures for somatic and psychiatric in- and outpatient services, purchase of prescription medications and paid assistance at home were obtained from national registries for the years 2002-2017. RESULTS: We included 412 in the IRD cohort (6,290 person years) and 1656 (25,088 person years) in the control cohort. Average, annual health care expenditures from age 0-48 years of age were €1,488 (SD 4,711) in the IRD cohort and €1,030 (4,639) in the control cohort. The largest difference was for out-patient eye care (13.26 times greater, 95% confidence interval 12.90-13.64). Psychiatric in-patient expenditures were 1.71 times greater (95% CI 1.66-1.76) in the IRD cohort but psychiatric out-patient health care costs were comparable between groups. CONCLUSIONS: Health care costs were approximately 40% greater in the IRD cohort compared to an age- and sex-matched sample from the general Danish population. This is relevant in the current situation with a number of trials aimed at treating IRDs using genetically based therapies. Although eye care expenditures were many times greater, they made up < 10% of the total health care expenditures even in the IRD cohort. The reduced costs related to injuries in the visually impaired cohort was a surprising finding but may reflect a reduced propensity to seek medical care rather than a reduced risk of injuries.

A novel compound heterozygous BEST1 gene mutation in two siblings causing autosomal recessive bestrophinopathy.

PURPOSE: To describe the clinical features, imaging characteristics, and genetic test results associated with a novel compound heterozygous mutation of the BEST1 gene in two siblings with autosomal recessive bestrophinopathy. METHODS: Two siblings underwent a complete ophthalmic examination, including dilated fundus examination, fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, fluorescein angiography, electroretinography, and electrooculography. A clinical diagnosis of autosomal recessive bestrophinopathy was established based on ocular examination and multimodal retinal imaging. Subsequently, clinical exome sequencing consisting of a panel of 6670 genes was carried out to confirm the diagnosis and assess genetic alterations in the protein-coding region of the genome of the patients. The identified mutations were tested in the two affected siblings and one of their parents. RESULTS: Two siblings (a 17-year-old female and a 15-year-old male) presented with reduced visual acuity and bilaterally symmetrical subretinal deposits of hyperautofluorescent materials in the posterior pole, which showed staining in the late phase of fluorescein angiogram. Spectral-domain optical coherence tomography demonstrated hyperreflective subretinal deposits and subretinal fluid accumulation. Both patients shared two mutations in the protein-coding region of the BEST1 gene, c.103G > A, p.(Glu35Lys) and c.313C > A, p.(Arg105Ser) (a novel disease-causing mutation). Sanger sequencing confirmed that the unaffected mother of the proband was carrying p.(Glu35Lys) variant in a heterozygous state. CONCLUSIONS: We have identified and described the phenotype of a novel disease-causing mutation NM_004183.4:c.313C > A, p.(Arg105Ser) in a heterozygous state along with a previously reported mutation NM_004183.4:c.103G > A, p.(Glu35Lys) of the BEST1 gene in two related patients with autosomal recessive bestrophinopathy.

Whole Genome Sequencing, Focused Assays and Functional Studies Increasing Understanding in Cryptic Inherited Retinal Dystrophies.

The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy. In this study, we investigated whole genome sequencing (WGS), focused assays and functional studies to assist with unsolved IRD cases and facilitate integration of these approaches to a broad molecular diagnostic clinical service. The WGS approach identified variants not covered or underinvestigated by targeted capture panel-based clinical testing strategies in six families. This included structural variants, with notable benefit of the WGS approach in repetitive regions demonstrated by a family with a hybrid gene and hemizygous missense variant involving the opsin genes, OPN1LW and OPN1MW. There was also benefit in investigation of the repetitive GC-rich ORF15 region of RPGR. Further molecular investigations were facilitated by focused assays in these regions. Deep intronic variants were identified in IQCB1 and ABCA4, with functional RNA based studies of the IQCB1 variant revealing activation of a cryptic splice acceptor site. While targeted capture panel-based methods are successful in achieving an efficient molecular diagnosis in a proportion of cases, this study highlights the additional benefit and clinical value that may be derived from WGS, focused assays and functional genomics in the highly heterogeneous IRDs.

The Clinical Spectrum and Disease Course of DRAM2 Retinopathy.

Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone-rod or rod-cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.

[Inherited retinal dystrophy: first results of RPE65 gene replacement therapy in Russia]. / Nasledstvennaya distrofiya setchatki: pervye rezul'taty posle RPE65-genozamestitel'noi terapii v Rossii.

PURPOSE: To present the main aspects of interdisciplinary diagnostics of patients with hereditary retinal diseases and the first results of the follow-up of patients with inherited retinal dystrophies (IRD) caused by biallelic mutations in the gene RPE65 after gene replacement therapy in Russia. MATERIAL AND METHODS: The cohort of patients consisted of six children (5-15 years old) with the diagnosis of Leber amaurosis type 2. All patients underwent a multi-disciplinary examination using conventional clinical, instrumental and molecular-genetic methods. Genetic diagnosis was established based on the results of two-stage DNA diagnostics using high-performance parallel sequencing of a custom panel and family segregation analysis by Sanger sequencing. RESULTS: In the Research Centre for Medical Genetics the first group of Russian patients with an orphan inherited retinal disease was verified, they underwent subretinal injection of the gene replacement drug Voretigene neparvovec (12 eyes) in the Helmholtz National Medical Research Center of Eye Diseases. According to the regulated terms of monitoring gene therapy patients, they were examined in the Research Centre for Medical Genetics after 1, 3, 6 and 12 months, and then once per year. Thus, the available data allows us to analyze the first results 3 months after the treatment. CONCLUSION: The presented data on inherited retinal dystrophies caused by biallelic mutations in the RPE65 gene emphasize the need to change the diagnostic algorithm in the ophthalmic practice. The use of clinical instrumental and molecular genetic diagnostic methods makes it possible to apply etiotropic treatment to patients with a disabling disease that was previously considered untreatable. The gene replacement drug Voretigene neparvovec registered in Russia showed irrefutable first positive results in all targeted patients.