Characterisation and quantification of F8 transcripts of ten putative splice site mutations

2015 ◽  
Vol 113 (03) ◽  
pp. 585-592 ◽  
Author(s):  
Yeling Lu ◽  
Yufeng Ruan ◽  
Qiulan Ding ◽  
Xuefeng Wang ◽  
Xiaodong Xi ◽  
...  
Keyword(s):  
Splice Site ◽  
Mrna Level ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fluorescent Pcr ◽  
Cryptic Splice Sites

SummaryMutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

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.

scholarly journals The Allele-Specific Suppressor sup-39 Alters Use of Cryptic Splice Sites in Caenorhabditis elegans

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1169-1179
Author(s):  
A Brock Roller ◽  
David C Hoffman ◽  
Alan M Zahler
Keyword(s):  
Caenorhabditis Elegans ◽  
Splice Site ◽  
Consensus Sequence ◽  
Splice Junction ◽  
Splice Sites ◽  
Wild Type ◽  
Splice Donor ◽  
Nonsense Mediated Decay ◽  
Allele Specific ◽  
Cryptic Splice Sites

Abstract Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5′ end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5′ splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

scholarly journals Activation of Cryptic 3′ Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 696 ◽  
Author(s):  
Heegyum Moon ◽  
Ha Na Jang ◽  
Yongchao Liu ◽  
Namjeong Choi ◽  
Jagyeong Oh ◽  
...  
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Exon Skipping ◽  
Cassette Exon ◽  
Intron Splicing ◽  
Splice Sites ◽  
Polypyrimidine Tract ◽  
Associated Sequences ◽  
Cryptic Splice Sites ◽  
Survival Of Motor Neuron

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3′ splice-site (3′AG′) usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3′AG′ (3′AG′1), its associated branch point (BP′) and polypyrimidine tract (PPT′) sequences directs SRSF2 to promote a second 3′AG′ (3′AG′2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3′AG′1 and 3′AG′2 and their associated sequences triggered usage of a third 3′AG′3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3′AG′ cryptic splice-sites, intron splicing from the canonical 3′AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3′AG′ activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3′AG′ and inhibiting downstream intron splicing.

scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

scholarly journals Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA

1999 ◽  
Vol 65 (1) ◽  
pp. 322-326 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferre-Aubineau ◽  
Berangere Mignotte ◽  
Berthe Marie Imbert-Marcille ◽  
Sylviane Billaudel
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Reproducible Method ◽  
Dna Enzyme Immunoassay

ABSTRACT To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 104 to 107 copies of HAV/gram or 400 to 106 50% tissue culture infective doses/ml.

scholarly journals Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

2002 ◽  
Vol 68 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
Camile Pizeta Semighini ◽  
Mozart Marins ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Quantitative Analysis ◽  
Real Time ◽  
Aspergillus Nidulans ◽  
Abc Transporter ◽  
Reverse Transcription ◽  
Wild Type ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Reverse Transcription Pcr ◽  
Nitroquinoline Oxide

ABSTRACT The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

scholarly journals Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

1994 ◽  
Vol 14 (5) ◽  
pp. 3426-3433 ◽  
Author(s):  
B Carr ◽  
P Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Splice Site ◽  
General Characteristic ◽  
Chain Gene ◽  
Splice Sites ◽  
Wild Type ◽  
Reading Frame ◽  
Intron Splice ◽  
Mature Mrna ◽  
Imprecise Excision

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

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