scholarly journals X-Linked Retinitis Pigmentosa Caused by Non-Canonical Splice Site Variants in RPGR

2021 ◽  
Vol 22 (2) ◽  
pp. 850
Author(s):  
Friederike Kortüm ◽  
Sinja Kieninger ◽  
Pascale Mazzola ◽  
Susanne Kohl ◽  
Bernd Wissinger ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Splice Site ◽  
Exon Skipping ◽  
Open Reading Frame ◽  
Aberrant Splicing ◽  
Reading Frame ◽  
Pathogenic Variants ◽  
Nucleotide Addition ◽  
Intron 2

We aimed to validate the effect of non-canonical splice site variants in the RPGR gene in five patients from four families diagnosed with retinitis pigmentosa. Four variants located in intron 2 (c.154 + 3_154 + 6del), intron 3 (c.247 + 5G>A), intron 7 (c.779-5T>G), and intron 13 (c.1573-12A>G), respectively, were analyzed by means of in vitro splice assays. Splicing analysis revealed different aberrant splicing events, including exon skipping and intronic nucleotide addition, which are predicted to lead either to an in-frame deletion affecting relevant protein domains or to a frameshift of the open reading frame. Our data expand the landscape of pathogenic variants in RPGR, thereby increasing the genetic diagnostic rate in retinitis pigmentosa and allowing patients harboring the analyzed variants to be enrolled in clinical trials.

scholarly journals Evaluation of suspected autosomal Alport Syndrome synonymous variants

Kidney360 ◽  
2021 ◽  
pp. 10.34067/KID.0005252021
Author(s):  
Rini Rossanti ◽  
Tomoko Horinouchi ◽  
Tomohiko Yamamura ◽  
China Nagano ◽  
Nana Sakakibara ◽  
...  
Keyword(s):  
Alport Syndrome ◽  
Autosomal Dominant ◽  
Exon Skipping ◽  
Aberrant Splicing ◽  
Synonymous Mutations ◽  
Pathogenic Variants ◽  
Functional Consequences ◽  
In Vitro Splicing

【Background】 Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in autosomal Alport syndrome cases, synonymous mutations in these genes have rarely been identified. 【Methods】 We conducted in silico splicing analysis using the Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of 4 synonymous variants detected in 4 patients with suspected autosomal dominant Alport syndrome (COL4A3 (c.693G>A (p.Val231=) and COL4A4 (c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=))). 【Results】 Both in vivo and in vitro splicing assays showed exon skipping in 2 out of the 4 synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using the HSF and Alamut suggested that these 2 variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other 2 variants did not induce aberrant splicing. 【Conclusions】 This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing as shown in 2 of our cases. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in autosomal dominant Alport syndrome cases.

scholarly journals Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 378 ◽  
Author(s):  
Vasileios Toulis ◽  
Vianney Cortés-González ◽  
Marta de Castro-Miró ◽  
Juliana Ferraz Sallum ◽  
Jaume Català-Mora ◽  
...  
Keyword(s):  
Diagnostic Yield ◽  
Intron Retention ◽  
Retinal Dystrophy ◽  
Genetic Diagnosis ◽  
Exon Skipping ◽  
Cell Therapies ◽  
Reading Frame ◽  
Pathogenic Variants

Aims: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). Methods: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. Results: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. Conclusions: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype–phenotype correlations and allow patients to opt for the emerging gene and cell therapies.

scholarly journals Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants

2021 ◽  
Vol 22 (10) ◽  
pp. 5396
Author(s):  
Nicole Weisschuh ◽  
Pascale Mazzola ◽  
Miriam Bertrand ◽  
Tobias B. Haack ◽  
Bernd Wissinger ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Splice Site ◽  
Age Of Onset ◽  
Splice Variants ◽  
Exon Skipping ◽  
Cone Dystrophy ◽  
Compound Heterozygous ◽  
Pathogenic Variants ◽  
Splice Site Variant

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep

2008 ◽  
Vol 89 (7) ◽  
pp. 1699-1708 ◽  
Author(s):  
Hong Li ◽  
Cristina W. Cunha ◽  
Christopher J. Davies ◽  
Katherine L. Gailbreath ◽  
Donald P. Knowles ◽  
...  
Keyword(s):  
Lymphoproliferative Disease ◽  
Infected Sheep ◽  
Open Reading Frame ◽  
Malignant Catarrhal Fever ◽  
Defence Mechanism ◽  
Reading Frame ◽  
Initial Infection ◽  
Nasal Secretions ◽  
Replication Site

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (∼7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

2015 ◽  
Vol 23 (8) ◽  
pp. 1341-1348 ◽  
Author(s):  
Ngoc B Lu-Nguyen ◽  
Susan A Jarmin ◽  
Amer F Saleh ◽  
Linda Popplewell ◽  
Michael J Gait ◽  
...  
Keyword(s):  
Exon Skipping ◽  
Open Reading Frame ◽  
Mdx Mice ◽  
Reading Frame

scholarly journals Design of in vitro Transcribed mRNA Vectors for Research and Therapy

2019 ◽  
Vol 73 (5) ◽  
pp. 391-394
Author(s):  
Marina Tusup ◽  
Lars E. French ◽  
Mara De Matos ◽  
David Gatfield ◽  
Thomas Kundig ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Open Reading Frame ◽  
Purification Method ◽  
Reading Frame ◽  
Mrna Purification ◽  
Functional Regions ◽  
In Cells

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

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