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      Expanding the phenotypic spectrum in RDH12-associated retinal disease

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          Abstract

          Retinol dehydrogenase 12, RDH12, plays a pivotal role in the visual cycle to ensure the maintenance of normal vision. Alterations in activity of this protein result in photoreceptor death and decreased vision beginning at an early age and progressing to substantial vision loss later in life. Here we describe 11 patients with retinal degeneration that underwent next-generation sequencing (NGS) with a targeted panel of all currently known inherited retinal degeneration (IRD) genes and whole-exome sequencing to identify the genetic causality of their retinal disease. These patients display a range of phenotypic severity prompting clinical diagnoses of macular dystrophy, cone-rod dystrophy, retinitis pigmentosa, and early-onset severe retinal dystrophy all attributed to biallelic recessive mutations in RDH12. We report 15 causal alleles and expand the repertoire of known RDH12 mutations with four novel variants: c.215A > G (p.Asp72Gly); c.362T > C (p.Ile121Thr); c.440A > C (p.Asn147Thr); and c.697G > A (p.Val233Ille). The broad phenotypic spectrum observed with biallelic RDH12 mutations has been observed in other genetic forms of IRDs, but the diversity is particularly notable here given the prior association of RDH12 primarily with severe early-onset disease. This breadth emphasizes the importance of broad genetic testing for inherited retinal disorders and extends the pool of individuals who may benefit from imminent gene-targeted therapies.

          Most cited references22

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          Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing.

          Next-generation sequencing-based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques with regard to test accuracy and reproducibility have not been fully defined. We developed a targeted enrichment and next-generation sequencing approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy, and glaucoma. In preparation for providing this genetic eye disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, and reproducibility, as well as the clinical sensitivity, of the test. The GEDi test is highly reproducible and accurate, with sensitivity and specificity of 97.9 and 100%, respectively, for single-nucleotide variant detection. The sensitivity for variant detection was notably better than the 88.3% achieved by whole-exome sequencing using the same metrics, because of better coverage of targeted genes in the GEDi test as compared with a commercially available exome capture set. Prospective testing of 192 patients with inherited retinal degenerations indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Based on quantified performance metrics, the data suggest that selective targeted enrichment is preferable to whole-exome sequencing for genetic diagnostic testing.
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            Critical residues for structure and catalysis in short-chain dehydrogenases/reductases.

            Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.
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              Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes

              Background The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease. Results We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3′ and 5′ alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products. Conclusions To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.
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                Author and article information

                Journal
                Cold Spring Harb Mol Case Stud
                Cold Spring Harb Mol Case Stud
                cshmcs
                cshmcs
                cshmcs
                Cold Spring Harbor Molecular Case Studies
                Cold Spring Harbor Laboratory Press
                2373-2873
                February 2020
                : 6
                : 1
                : a004754
                Affiliations
                [1 ]Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts 02114, USA;
                [2 ]Center for Mendelian Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA;
                [3 ]Retina Service, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts 02114, USA
                Author notes
                Author information
                http://orcid.org/0000-0001-7064-4934
                http://orcid.org/0000-0001-9439-8133
                http://orcid.org/0000-0002-9220-2261
                http://orcid.org/0000-0001-6809-9818
                Article
                MCS004754Sco
                10.1101/mcs.a004754
                6996522
                32014858
                e5e79526-0e30-4ed8-a583-b4524006822e
                © 2020 Scott et al.; Published by Cold Spring Harbor Laboratory Press

                This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

                History
                : 11 September 2019
                : 5 December 2019
                Page count
                Pages: 14
                Funding
                Funded by: National Eye Institute , open-funder-registry 10.13039/100000053;
                Award ID: R01EY012910
                Award ID: R01EY026904
                Award ID: P30EY014104
                Funded by: Foundation Fighting Blindness
                Award ID: EGI-GE-1218-0753-UCSD
                Funded by: National Human Genome Research Institute
                Funded by: National Eye Institute
                Funded by: National Heart
                Funded by: Lung and Blood Institute
                Award ID: UM1HG008900
                Funded by: National Human Genome Research Institute , open-funder-registry 10.13039/100000051;
                Award ID: R01 HG009141
                Funded by: Mukai Fund, Massachusetts Eye and Ear
                Categories
                Research Report

                central scotoma,cone-rod dystrophy,macular dystrophy,peripheral visual field loss,pigmentary retinal degeneration,progressive central visual loss,progressive visual field defects,severe visual impairment

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