High-Throughput Sequencing to Identify Mutations Associated with Retinal Dystrophies.
TL;DR: In this paper, a whole exome sequencing (WES) pipeline was established to rapidly identify disease-associated mutations in patients, and Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families.
Abstract: Retinal dystrophies (RD) are clinically and genetically heterogenous disorders showing mutations in over 270 disease-associated genes. Several millions of people worldwide are affected with different types of RD. Studying the relevance of disease-associated sequence alterations will assist in understanding disorders and may lead to the development of therapeutic approaches. Here, we established a whole exome sequencing (WES) pipeline to rapidly identify disease-associated mutations in patients. Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families. We analyzed 26 unrelated patients with different syndromic and non-syndromic clinical manifestations of RD. All patients underwent ophthalmic examinations. We identified nine novel disease-associated sequence variants among 37 variants identified in total. The sequence variants located to 17 different genes. Interestingly, two cases presenting with Stargardt disease carried deep-intronic variants in ABCA4. We have classified 21 variants as pathogenic variants, 4 as benign/likely benign variants, and 12 as variants of uncertain significance. This study highlights the importance of WES-based mutation analyses in RD patients supporting clinical decisions, broadly based genetic diagnosis and support genetic counselling. It is essential for any genetic therapy to expand the mutation spectrum, understand the genes’ function, and correlate phenotypes with genotypes.
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TL;DR: A detailed overview of the role of NR2E3 in normal development and disease, and associated clinical phenotypes, animal models, and therapeutic studies can be found in this paper .
Abstract: NR2E3 is a nuclear hormone receptor gene required for the correct development of the retinal rod photoreceptors. Expression of NR2E3 protein in rod cell precursors suppresses cone-specific gene expression and, in concert with other transcription factors including NRL, activates the expression of rod-specific genes. Pathogenic variants involving NR2E3 cause a spectrum of retinopathies, including enhanced S-cone syndrome, Goldmann–Favre syndrome, retinitis pigmentosa, and clumped pigmentary retinal degeneration, with limited evidence of genotype–phenotype correlations. A common feature of NR2E3-related disease is an abnormally high number of cone photoreceptors that are sensitive to short wavelength light, the S-cones. This characteristic has been supported by mouse studies, which have also revealed that loss of Nr2e3 function causes photoreceptors to develop as cells that are intermediate between rods and cones. While there is currently no available cure for NR2E3-related retinopathies, there are a number of emerging therapeutic strategies under investigation, including the use of viral gene therapy and gene editing, that have shown promise for the future treatment of patients with NR2E3 variants and other inherited retinal diseases. This review provides a detailed overview of the current understanding of the role of NR2E3 in normal development and disease, and the associated clinical phenotypes, animal models, and therapeutic studies.
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TL;DR: Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends thatclinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments–approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.
17,834 citations
TL;DR: This study investigated the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carrying RPE65 complementary DNA (cDNA) and found three patients with LCA2 had an acceptable local and systemic adverse-event profile after delivery of AAV2.hRPE65v2.
Abstract: S um m a r y Leber's congenital amaurosis (LCA) is a group of inherited blinding diseases with onset during childhood. One form of the disease, LCA2, is caused by mutations in the retinal pigment epithelium-specific 65-kDa protein gene (RPE65). We investiga t ed the safety of subretinal delivery of a recombinant adeno-associated virus (AAV) carry- ing RPE65 complementary DNA (cDNA) (ClinicalTrials.gov number, NCT00516477). Three patients with LCA2 had an acceptable local and systemic adverse-event pro- file after delivery of AAV2.hRPE65v2. Each patient had a modest improvement in measures of retinal function on subjective tests of visual acuity. In one patient, an asymptomatic macular hole developed, and although the occurrence was considered to be an adverse event, the patient had some return of retinal function. Although the follow-up was very short and normal vision was not achieved, this study pro- vides the basis for further gene therapy studies in patients with LCA.
2,066 citations
TL;DR: Three young adult patients with early-onset, severe retinal dystrophy were administered subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human R PE65 promoter.
Abstract: Early-onset, severe retinal dystrophy caused by mutations in the gene encoding reti- nal pigment epithelium-specific 65-kDa protein (RPE65) is associated with poor vi- sion at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Gold- mann perimetry in any of the three patients. We detected no change in retinal re- sponses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747.)
1,912 citations
TL;DR: An overview of the evolution of NGS is provided and the most significant improvements in sequencing technologies and library preparation protocols are discussed and the current landscape of N GS applications is explored to provide a perspective for future developments.
1,342 citations
TL;DR: Voretigene neparvovec gene replacement improved functional vision in RPE65-mediated inherited retinal dystrophy previously medically untreatable.
1,081 citations