Mutational analysis of ectopic factor VIII transcripts from hemophilia A patients: identification of cryptic splice site, exon skipping and novel point mutations

1997 ◽  
Vol 100 (5-6) ◽  
pp. 508-511 ◽  
Author(s):  
Kamiab Tavassoli ◽  
Antonin Eigel ◽  
Hartmut Pollmann ◽  
Jürgen Horst
Keyword(s):  
Factor Viii ◽  
Splice Site ◽  
Hemophilia A ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Exon Skipping ◽  
Cryptic Splice Site

scholarly journals Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3015-3020
Author(s):  
VR Arruda ◽  
WC Pieneman ◽  
PH Reitsma ◽  
PP Deutz-Terlouw ◽  
JM Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Southern Blotting ◽  
Hemophilia A ◽  
Severe Disease ◽  
Point Mutations ◽  
Electrophoretic Pattern ◽  
Telomeric Region ◽  
Factor Viii Gene ◽  
Single Strand Conformational Polymorphism ◽  
Almost All

The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene

Genomics ◽  
1988 ◽  
Vol 2 (1) ◽  
pp. 32-36 ◽  
Author(s):  
Hagop Youssoufian ◽  
Haig H. Kazazian ◽  
Achyut Patel ◽  
Sophia Aronis ◽  
George Tsiftis ◽  
...  
Keyword(s):  
Factor Viii ◽  
Splice Site ◽  
Hemophilia A ◽  
Splice Site Mutation ◽  
Donor Splice Site ◽  
Factor Viii Gene ◽  
Site Mutation ◽  
Donor Splice ◽  
Intron 4

scholarly journals Eleven novel mutations in the factor VIII gene from Brazilian hemophilia A patients

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3015-3020 ◽  
Author(s):  
VR Arruda ◽  
WC Pieneman ◽  
PH Reitsma ◽  
PP Deutz-Terlouw ◽  
JM Annichino-Bizzacchi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Southern Blotting ◽  
Hemophilia A ◽  
Severe Disease ◽  
Point Mutations ◽  
Electrophoretic Pattern ◽  
Telomeric Region ◽  
Factor Viii Gene ◽  
Single Strand Conformational Polymorphism ◽  
Almost All

Abstract The molecular characterization of the mutations in hemophilia A patients is hampered by the large size of the factor VIII gene and the great heterogeneity of mutations. In this study, we have performed a protocol involving multiplex polymerase chain reaction in which 19 exons were amplified in four different combinations followed by nonradioactive single-strand conformational polymorphism (SSCP) to screen for mutations. Southern blotting was used to detect inversion of the factor VIII gene resulting from recombination between copies of the gene A (F8A) located in intron 22 of the factor VIII gene and two copies close telomeric region of X chromosome. Forty-two hemophilia A patients (21 with severe and 21 with mild-to-moderate disease) were studied. The inversion of factor VIII occurred in 13 of 21 patients affected by severe hemophilia A. One patient showed a large extra band in addition to the three bands observed after Southern blotting with the F8A probe. An abnormal electrophoretic pattern of SSCP was detected in 85% and 50% of the patients affected by mild-to-moderate and severe disease, respectively. Sixteen different mutations were identified. Eleven mutations were novel and comprised 9 point mutations and 2 small deletions. This study shows that the methodology used is safe and rapid and has potential for detecting almost all of the genetic defects of the studied hemophilia A patients.

scholarly journals Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 526-534 ◽  
Author(s):  
Ali Ramezani ◽  
Robert G. Hawley
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Insertional Mutagenesis ◽  
Hemophilia A ◽  
Point Mutations ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Human Factor Viii ◽  
A1 Domain

Abstract Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A.

Targeted DNA- and RNA-based next-generation sequencing for identifying MET exon 14 alterations in pulmonary sarcomatoid carcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20591-e20591
Author(s):  
Jianming Ying ◽  
Lianju Gao ◽  
Yan Li ◽  
Bing Liu ◽  
Lamei Deng ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Splice Site ◽  
Point Mutations ◽  
Sarcomatoid Carcinoma ◽  
Exon Skipping ◽  
Performance Comparison ◽  
Next Generation ◽  
Dna And Rna ◽  
Pulmonary Sarcomatoid Carcinoma ◽  
Generation Sequencing

e20591 Background:Highly diverse somatic splice site alteration at MET exon 14 ( METex14) result in exon skipping, which is supposed to be a therapeutic target in NSCLC. Here we report detection of METex14 alterations using targeted DNA- and RNA-based Next-Generation Sequencing (NGS) in pulmonary sarcomatoid carcinoma (PSC) with a high frequency of METex14 skipping. Methods: Tumor specimens were collected from 100 Chinese PSC patients. DNA and RNA were subject to targeted NGS, allowing the detection of somatic splice site alterations and intragenic METex14 skipping respectively. Then, somatic mutations that lead to METex14 skipping were recognized. Meanwhile, cross-platform performance comparison for detecting MET exon 14 skipping was achieved. Moreover, Sanger sequencing was also performed on the METex14-positive specimens. Results: So far, we have detected genetic aberrations in 34 FFPE samples. For RNA-based NGS, METex14 skipping was identified in 8 (23.5%) of 34 patient cases. The population frequency was accordant to the reported percent of 22% (PMID: 26215952). And 5 (62.5%) of 8 METex14-positive patients were detected somatic splice site mutations by DNA-based NGS, including 4 cases with known point mutations at the 3’ splice region and one case with a novel deletion (chr7: 116412027- 116412042) at MET exon14 region, which is newly discovered. No somatic mutation was found at the splice junctions of METex14 in the remaining 3 samples using DNA-based NGS. Conclusions: Mutational events of MET leading to exon 14 skipping are frequent occurred in Chinese PSC patients. Our study also suggests that RNA-based NGS could identify more METex14 skipping, and thus provide more accurate results than DNA-based NGS.

scholarly journals Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicity in vitro

2019 ◽  
Vol 6 (8) ◽  
pp. 191128 ◽  
Author(s):  
Jonathan Cook ◽  
Elizabeth de Wolf ◽  
Nicholas Dale
Keyword(s):  
Alternative Splicing ◽  
Branch Point ◽  
Splice Site ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Future Research ◽  
Cryptic Splice Site ◽  
Toxicity In Vitro

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

scholarly journals Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

1996 ◽  
Vol 16 (6) ◽  
pp. 3012-3022 ◽  
Author(s):  
D Y Hwang ◽  
J B Cohen
Keyword(s):  
Splice Site ◽  
Point Mutations ◽  
Exon Skipping ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Enhancer Activity ◽  
S1 Nuclease ◽  
Exon Definition ◽  
Small Nuclear Rnas

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.

scholarly journals Ab initio prediction of mutation-induced cryptic splice-site activation and exon skipping

2009 ◽  
Vol 17 (6) ◽  
pp. 759-765 ◽  
Author(s):  
Petr Divina ◽  
Andrea Kvitkovicova ◽  
Emanuele Buratti ◽  
Igor Vorechovsky
Keyword(s):  
Ab Initio ◽  
Splice Site ◽  
Exon Skipping ◽  
Cryptic Splice Site ◽  
Ab Initio Prediction

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease

2006 ◽  
Vol 96 (12) ◽  
pp. 711-716 ◽  
Author(s):  
Lisa Gallinaro ◽  
Francesca Sartorello ◽  
Elena Pontara ◽  
Maria Cattini ◽  
Antonella Bertomoro ◽  
...  
Keyword(s):  
Splice Site ◽  
Stop Codon ◽  
Consensus Sequence ◽  
Premature Stop Codon ◽  
Exon Skipping ◽  
Von Willebrand Disease ◽  
Cryptic Splice Site ◽  
Recessive Type ◽  
Von Willebrand

SummaryWe describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534–3C>A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon inVWF propeptide, so the probandVWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534–3C>A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3VWD. This mutation therefore appears to be peculiar to type 1 VWD.

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