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 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

Engineered I-CreI Derivatives Cleaving Sequences from the Human XPC Gene can Induce Highly Efficient Gene Correction in Mammalian Cells

2007 ◽  
Vol 371 (1) ◽  
pp. 49-65 ◽  
Author(s):  
Sylvain Arnould ◽  
Christophe Perez ◽  
Jean-Pierre Cabaniols ◽  
Julianne Smith ◽  
Agnès Gouble ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Correction ◽  
Highly Efficient ◽  
Efficient Gene
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