scholarly journals Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 363 ◽  
Author(s):  
Sarah Naessens ◽  
Laurien Ruysschaert ◽  
Steve Lefever ◽  
Frauke Coppieters ◽  
Elfride De Baere
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Quantitative Polymerase Chain Reaction ◽  
Dominant Negative ◽  
Locked Nucleic Acid ◽  
Dominant Negative Effect ◽  
Acid Modification ◽  
Autosomal Dominant Retinitis Pigmentosa ◽  
Knock Down ◽  
Negative Effect

The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%–2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal disease (IRD). The mutant NR2E3 protein has a presumed dominant negative effect (DNE) by competition for dimer formation with Cone-Rod Homeobox (CRX) but with abolishment of DNA binding, acting as a repressor in trans. Both the frequency and DNE of G56R make it an interesting target for allele-specific knock-down of the mutant allele using antisense oligonucleotides (AONs), an emerging therapeutic strategy for IRD. Here, we designed gapmer AONs with or without a locked nucleic acid modification at the site of the mutation, which were analyzed for potential off-target effects. Next, we overexpressed wild type (WT) or mutant NR2E3 in RPE-1 cells, followed by AON treatment. Transcript and protein levels of WT and mutant NR2E3 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot respectively. All AONs showed a general knock-down of mutant and WT NR2E3 on RNA and protein level, showing the accessibility of the region for AON-induced knockdown. Further modifications are needed however to increase allele-specificity. In conclusion, we propose the first proof-of-concept for AON-mediated silencing of a single nucleotide variation with a dominant negative effect as a therapeutic approach for NR2E3-associated adRP.

scholarly journals Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa

2019 ◽  
Vol 20 (10) ◽  
pp. 2542 ◽  
Author(s):  
Michalitsa Diakatou ◽  
Gaël Manes ◽  
Beatrice Bocquet ◽  
Isabelle Meunier ◽  
Vasiliki Kalatzis
Keyword(s):  
Retinitis Pigmentosa ◽  
Genome Editing ◽  
Mutant Allele ◽  
Autosomal Dominant ◽  
Dominant Negative ◽  
Therapeutic Approaches ◽  
Autosomal Dominant Retinitis Pigmentosa ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Type Gene

Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to (i) invalidate both alleles, followed by supplementation of the wild type gene, (ii) specifically invalidate the mutant allele, with or without gene supplementation, or (iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.

scholarly journals The Role of Subunit Assembly in Peripherin-2 Targeting to Rod Photoreceptor Disk Membranes and Retinitis Pigmentosa

2003 ◽  
Vol 14 (8) ◽  
pp. 3400-3413 ◽  
Author(s):  
Christopher J.R. Loewen ◽  
Orson L. Moritz ◽  
Beatrice M. Tam ◽  
David S. Papermaster ◽  
Robert S. Molday
Keyword(s):  
Retinitis Pigmentosa ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Rod Photoreceptor ◽  
Wild Type ◽  
Photoreceptor Outer Segment ◽  
Negative Effect ◽  
Retinal Degenerative Diseases ◽  
Green Fluorescent

Peripherin-2 is a member of the tetraspanin family of membrane proteins that plays a critical role in photoreceptor outer segment disk morphogenesis. Mutations in peripherin-2 are responsible for various retinal degenerative diseases including autosomal dominant retinitis pigmentosa (ADRP). To identify determinants required for peripherin-2 targeting to disk membranes and elucidate mechanisms underlying ADRP, we have generated transgenic Xenopus tadpoles expressing wild-type and ADRP-linked peripherin-2 mutants as green fluorescent fusion proteins in rod photoreceptors. Wild-type peripherin-2 and P216L and C150S mutants, which assemble as tetramers, targeted to disk membranes as visualized by confocal and electron microscopy. In contrast the C214S and L185P mutants, which form homodimers, but not tetramers, were retained in the rod inner segment. Only the P216L disease mutant induced photoreceptor degeneration. These results indicate that tetramerization is required for peripherin-2 targeting and incorporation into disk membranes. Tetramerization-defective mutants cause ADRP through a deficiency in wild-type peripherin-2, whereas tetramerization-competent P216L peripherin-2 causes ADRP through a dominant negative effect, possibly arising from the introduction of a new oligosaccharide chain that destabilizes disks. Our results further indicate that a checkpoint between the photoreceptor inner and outer segments allows only correctly assembled peripherin-2 tetramers to be incorporated into nascent disk membranes.

scholarly journals SNRNP200 Mutations Cause Autosomal Dominant Retinitis Pigmentosa

2021 ◽  
Vol 7 ◽  
Author(s):  
Tao Zhang ◽  
Jingshan Bai ◽  
Xinyi Zhang ◽  
Xiaowei Zheng ◽  
Nan Lu ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Causative Mutation ◽  
Chinese Family ◽  
Zebrafish Embryos ◽  
Gene Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Negative Effect ◽  
In The Wild

The small nuclear ribonucleoprotein 200 kDa (SNRNP200) gene plays a key role in the maturation of pre-message RNA (pre-mRNA) splicing with the indication for the etiology of retinitis pigmentosa (RP). Gene recognition can facilitate the diagnosis of these patients for better clinical management, treatment and counseling. This study aimed to outline the causative mutation in a Chinese family and the pathogenic mechanism of this SNRNP200 mutation in RP. Eighteen individuals from the affected family underwent a complete ophthalmic examination. Whole exome sequencing (WES) was conducted to identify the pathogenic variant in the proband, which was then confirmed by Sanger sequencing. Expression of the SNRNP200 transcript in zebrafish was identified via whole mount in situ hybridization. Morpholino oligonucleotide (MO) and SNRNP200 wild and mutant mRNA were injected into zebrafish embryos followed by analyses of the systemic changes and retinal phenotypes using immunofluorescence. Heterozygous SNRNP200c.C6088T (p.Arg2030Cys) mutation was ascertained in two members of this family: the proband and his father (II-2). Overexpression of SNRNP200Arg2030Cys, but not SNRNP200WT caused systemic deformities in the wild-type zebrafish embryos with the retina primarily injured, and significantly increased death rates in the morphant embryos, in which the orthologous zebrafish SNRNP200 gene was blocked. In conclusion, this study reports a novel heterozygous SNRNP200c.C6088T mutation, which is evidenced to cause RP via a dominant-negative effect.

scholarly journals Homozygote loss-of-function variants in the human COCH gene underlie hearing loss

2020 ◽  
Author(s):  
Nada Danial-Farran ◽  
Elena Chervinsky ◽  
Prathamesh Thangaraj Nadar-Ponniah ◽  
Eran Cohen Barak ◽  
Shahar Taiber ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Autosomal Dominant ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Loss Of Function ◽  
Wild Type ◽  
Cos7 Cell ◽  
Gene Encoding ◽  
Negative Effect ◽  
Syndromic Hearing Loss

AbstractSince 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While an additional recessive variant in the COCH gene (DFNB110) has recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data has a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

scholarly journals A Dominant Negative Effect of eth-1r, a Mutant Allele of the Neurospora crassa S-Adenosylmethionine Synthetase-Encoding Gene Conferring Resistance to the Methionine Toxic Analogue Ethionine

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1455-1462
Author(s):  
José L Barra ◽  
Mario R Mautino ◽  
Alberto L Rosa
Keyword(s):  
Neurospora Crassa ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Resistant Phenotype ◽  
Negative Effect ◽  
Encoding Gene ◽  
Adenosylmethionine Synthetase ◽  
Adomet Synthetase ◽  
Identical Gene

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1  +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1  +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1  + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1  +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1  +/eth-1r AdoMet synthetase molecules.

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