Identification of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency

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.

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

Royal Society Open Science ◽

10.1098/rsos.191128 ◽

2019 ◽

Vol 6 (8) ◽

pp. 191128 ◽

Cited By ~ 3

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.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.3012 ◽

1996 ◽

Vol 16 (6) ◽

pp. 3012-3022 ◽

Cited By ~ 13

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.

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

European Journal of Human Genetics ◽

10.1038/ejhg.2008.257 ◽

2009 ◽

Vol 17 (6) ◽

pp. 759-765 ◽

Cited By ~ 42

Author(s):

Petr Divina ◽

Andrea Kvitkovicova ◽

Emanuele Buratti ◽

Igor Vorechovsky

Keyword(s):

Ab Initio ◽

Splice Site ◽

Exon Skipping ◽

Cryptic Splice Site ◽

Ab Initio Prediction

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

Thrombosis and Haemostasis ◽

10.1160/th06-07-0417 ◽

2006 ◽

Vol 96 (12) ◽

pp. 711-716 ◽

Cited By ~ 13

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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Background splicing and genetic disease

10.21203/rs.3.rs-92665/v1 ◽

2020 ◽

Author(s):

Diana Alexieva ◽

Yi Long ◽

Rupa Sarkar ◽

Hansraj Dhayan ◽

Emmanuel Bruet ◽

...

Keyword(s):

Splice Site ◽

Genetic Disease ◽

Exon Skipping ◽

Splice Sites ◽

Cryptic Splice Site ◽

Aberrant Splicing ◽

Low Level ◽

Splicing Mutations ◽

Brca1 Brca2 ◽

Rna Splice Sites

Abstract We report that low level background splicing by normal genes can be used to predict the likely effect of splicing mutations upon cryptic splice site activation and exon skipping, with emphasis on the DBASS databases, BRCA1, BRCA2 and DMD. In addition we show that background RNA splice sites are also involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer. We discuss how background splicing information might inform splicing therapy.

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Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650386 ◽

1996 ◽

Vol 75 (06) ◽

pp. 870-876 ◽

Cited By ~ 4

Author(s):

José Manuel Soria ◽

Lutz-Peter Berg ◽

Jordi Fontcuberta ◽

Vijay V Kakkar ◽

Xavier Estivill ◽

...

Keyword(s):

Splice Site ◽

Protein C ◽

Stop Codon ◽

Premature Stop Codon ◽

Allelic Exclusion ◽

Polymorphism Analysis ◽

Type I ◽

Protein C Deficiency ◽

Nonsense Mutations ◽

Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

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FAMILIAL GATA2 HAPLOINSUFFICIENCY CAUSED BY A NOVEL MUTATION INTRODUCING A CRYPTIC SPLICE SITE IN GATA2 EXON 3

10.26226/morressier.57bc1756d462b80290b4d80d ◽

2016 ◽

Author(s):

Claudia Wehr

Keyword(s):

Splice Site ◽

Novel Mutation ◽

Cryptic Splice Site

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Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽

10.1093/genetics/164.4.1345 ◽

2003 ◽

Vol 164 (4) ◽

pp. 1345-1353

Author(s):

Amber K Bowers ◽

Jennifer A Keller ◽

Susan K Dutcher

Keyword(s):

Molecular Markers ◽

Chlamydomonas Reinhardtii ◽

Candidate Genes ◽

Splice Site ◽

Genomic Dna ◽

Ribosomal Proteins ◽

Genomic Sequence ◽

Point Mutations ◽

Acceptor Site ◽

Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

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