scholarly journals Transposon clusters as substrates for aberrant splice-site activation

RNA Biology ◽  
2020 ◽  
pp. 1-14
Author(s):  
Maria Elena Vilar Alvarez ◽  
Martin Chivers ◽  
Ivana Borovska ◽  
Steven Monger ◽  
Eleni Giannoulatou ◽  
...  
Keyword(s):  
Splice Site ◽  
Aberrant Splice

Characterization of a Novel Aberrant Splice Site, 79bp Downstream of Exon 5 in the Human Factor 7 Gene Detected in Patients with Severe Congenital Factor VII Deficiency.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3481-3481
Author(s):  
Karin Wulff ◽  
Jan Astermark ◽  
Falko F H Herrmann ◽  
Günther Auerswald ◽  
Winnie Schröder
Keyword(s):  
Splice Site ◽  
Donor Splice Site ◽  
Aberrant Splice ◽  
The Novel ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Donor Splice ◽  
Naturally Occurring ◽  
Fvii Deficiency

Abstract Abstract 3481 Poster Board III-418 Hereditary FVII deficiency (FVIID) is a rare congenital bleeding disorder with an estimated prevalence of symptomatic individuals of 1:500,000. In the “Greifswald Registry FVII Deficiency” molecular defects of more than 1000 FVII deficient patients were described. By direct sequencing of the F7 genes in congenital FVIID revealed 146 different F7 gene mutations including 25 different mutations (18% of all) in the naturally-occurring acceptor or donor splice sites (Tab.1) were identified. In seven FVIID patients from Sweden and Germany the novel lesion g.IVS5+78G>A - downstream of the naturally-occurring donor splice sites of exon 5 - was identified. This variation was detected heterozygous in FVIID patients with FVII: C levels of 15%, 27%, 31%, 40% and 65%, and FVII: Ag levels between 25-50%. In two compound heterozygous patients with FVII: C levels of 1% und FVII: Ag levels of 2% and 3% respectively, one well-known causative FVII mutation is combined with the novel lesion g.IVS5+78G>A. The influence of this novel F7 gene variation on splicing was investigated by RT-PCR analysis and in vitro expression studies using exon-trap vector constructs. The total RNA was isolated from peripheral leucocytes and analyzed by one step RT-PCR and sequencing. Fragments of exon 5 and a part of the flanking intron 5 region (g.7679 –g.8073) were amplified of patients' DNA and cloned into the exon trap-vector pET01. Different vector constructs containing minigenes of the wild type (g.IVS5+78G) or mutant form (g.IVS5+78A) and the corresponding minigenes with an “optimized” naturally-occurring donor splice site in position +5 respectively were transfected into HEK293 cells. The expressed RNA was isolated and characterized. Consensus Values (CV) for all donor splice sites were calculated using a splice site detection tool according Shapiro and Senapathy (1987). The RT-PCR analysis in patients indicate that the novel variation g.IVS5+78G>A in intron 5 created an aberrant splice site in position 79bp downstream of exon 5 even though the naturally-occurring donor-splice-site of exon 5 is not abolished. An insertion of 79bp of intron 5 into the mRNA leads to a frame shift and predicts a premature termination 11 codons past the last unaltered codon. Minigenes include the naturally-occurring splice site and the variation g.IVS5+78A used exclusively the aberrant splice position 79bp downstream of exon 5 whereas wild type minigenes with the naturally-occurring splice site and the wild type form g.IVS5+78G produced normally spliced mRNA. In a following experiment the “naturally-occurring splice site” of exon 5 was optimized by the additional substitution g.IVS5+5C>G which increased their CV from 76.6 to 90.9 compared to the CV of the novel mutant g.IVS5+78A of 80.3. In presence of both mutations (g.IVS5+5G and g.IVS5+78A) only normal spliced mRNA was expressed of this minigene. In this construct the mutation g.IVS5+78G>A was without importance for the mRNA splicing. The results of the in vitro experiments demonstrated, that the Consensus Values (CV) seems to be an important factor for the selection of donor splice sites in the F7 gene. In the “Greifswald Registry FVII Deficiency” 26 different splice site variations in F7 gene were identified (Tab. 1). The atypical splice site variation g.IVS5+78G>A, +78bp downstream of exon 5 was present in 7 FVIID patients from Sweden and Germany in different genotypes. This novel F7 gene mutation g.IVS5+78G>A creates an aberrant splice site in position +79 of intron 5 and predicts premature termination. RNA analysis and expression studies demonstrated, that this novel F7 gene lesion is a type I mutation with low FVII:C and FVII: Ag levels and is the basis defect in 7 FVIID patients of the “Greifswald Registry FVII Deficiency”. Tab. 1 26 different intronic F7 gene mutations analysed in FVII deficiency patients of the “Greifswald Registry FVII Deficiency” Intron Acceptor splice site Intron Donor splice site 1b g.IVS1b-11G>A 1a g.IVS1a+5G>A 1b *g.IVS1b8del14bp 1a *g.IVS1a+6T>G 1b *g.IVS1b-3C>G 1a *g.IVS1a+8C>T 2 *g.IVS2-3C>G 2 g.IVS2+1G>A 3 g.IVS3-1G>A 2 *g.IVS2+1G>T 3 *g-IVS3-1G>T 2 *g.IVS2+1G>C 4 *g.IVS4-7T>G 2 *g.IVS2+1delG 7 *g.IVS7-10T>C 2 g.IVS2+5G>T 7 *g.IVS7-3C>G 3 *g.IVS3+1G>T 7 *g.IVS7-1G>A 4 g.IVS4+1G>A 5 *g.IVS5+78G>A 6 *g.IVS6+1G>A 6 g.IVS6+1G>T 6 *g.IVS6+3A>G 7 *g.IVS7+1G>A 7 g.IVS7+3_6 del4bp * novel mutations (HGMD Factor 7 Database, 2009 /http://www.hgmd.org) Disclosures: No relevant conflicts of interest to declare.

A novel aberrant splice site mutation in COL27A1 is responsible for Steel syndrome and extension of the phenotype to include hearing loss

2017 ◽  
Vol 173 (5) ◽  
pp. 1257-1263 ◽  
Author(s):  
Nesrin Gariballa ◽  
Afif Ben-Mahmoud ◽  
Makanko Komara ◽  
Aisha M. Al-Shamsi ◽  
Anne John ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Splice Site ◽  
Splice Site Mutation ◽  
Aberrant Splice ◽  
Site Mutation

scholarly journals 2'-O-Methoxyethyl Splice-Switching Oligos to Reverse Splicing from IVS2-745 β-Thalassemia Patient Cells: A Foundation for Potential Therapies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2244-2244
Author(s):  
Valentina Ghiaccio ◽  
Alisa Dong ◽  
Irene Motta ◽  
Shuling Guo ◽  
Raechel Peralta ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Splice Site ◽  
Globin Gene ◽  
Mrna Translation ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Globin Mrna ◽  
Aberrant Splicing ◽  
Advisory Committees ◽  
Intron 2

The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis. The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a cryptic 3' splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern. We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype. Moreover, we compared the effect of 2'-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2'-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia. In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence. Disclosures Dong: Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

scholarly journals An ancient germ cell-specific RNA-binding protein protects the germline from cryptic splice site poisoning

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ingrid Ehrmann ◽  
James H Crichton ◽  
Matthew R Gazzara ◽  
Katherine James ◽  
Yilei Liu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Splice Site ◽  
Cell Biology ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Aberrant Splice ◽  
Splice Site Selection ◽  
Ambient Concentrations ◽  
Splicing Patterns

Male germ cells of all placental mammals express an ancient nuclear RNA binding protein of unknown function called RBMXL2. Here we find that deletion of the retrogene encoding RBMXL2 blocks spermatogenesis. Transcriptome analyses of age-matched deletion mice show that RBMXL2 controls splicing patterns during meiosis. In particular, RBMXL2 represses the selection of aberrant splice sites and the insertion of cryptic and premature terminal exons. Our data suggest a Rbmxl2 retrogene has been conserved across mammals as part of a splicing control mechanism that is fundamentally important to germ cell biology. We propose that this mechanism is essential to meiosis because it buffers the high ambient concentrations of splicing activators, thereby preventing poisoning of key transcripts and disruption to gene expression by aberrant splice site selection.

scholarly journals Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

2019 ◽  
Vol 23 (12) ◽  
pp. 8046-8057 ◽  
Author(s):  
Zeyu Xiong ◽  
Yingjun Xie ◽  
Yi Yang ◽  
Yanting Xue ◽  
Ding Wang ◽  
...  
Keyword(s):  
Splice Site ◽  
Single Strand ◽  
Aberrant Splice ◽  
Gene Correction ◽  
Efficient Gene

scholarly journals Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

1994 ◽  
Vol 14 (11) ◽  
pp. 7445-7454 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Branch Point Sequence ◽  
Splice Site Sequence ◽  
Internal Exon ◽  
Cryptic Splice Sites

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

scholarly journals Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing

1994 ◽  
Vol 14 (11) ◽  
pp. 7445-7454
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Branch Point Sequence ◽  
Splice Site Sequence ◽  
Internal Exon ◽  
Cryptic Splice Sites

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

K120R mutation inactivates p53 by creating an aberrant splice site leading to nonsense-mediated mRNA decay

Oncogene ◽  
2018 ◽  
Vol 38 (10) ◽  
pp. 1597-1610 ◽  
Author(s):  
Seo-Young Lee ◽  
Jung-Hyun Park ◽  
Sangkyun Jeong ◽  
Bu-Yeo Kim ◽  
Yong-Kook Kang ◽  
...  
Keyword(s):  
Splice Site ◽  
Mrna Decay ◽  
Aberrant Splice ◽  
Nonsense Mediated Mrna Decay
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