Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events
Abstract Background Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targe...
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| Format: | Article |
| Language: | English |
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BMC
2017-11-01
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| Series: | Breast Cancer Research |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13058-017-0919-1 |
| _version_ | 1831539570459541504 |
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| author | Lucy C. de Jong Simone Cree Vanessa Lattimore George A. R. Wiggins Amanda B. Spurdle kConFab Investigators Allison Miller Martin A. Kennedy Logan C. Walker |
| author_facet | Lucy C. de Jong Simone Cree Vanessa Lattimore George A. R. Wiggins Amanda B. Spurdle kConFab Investigators Allison Miller Martin A. Kennedy Logan C. Walker |
| author_sort | Lucy C. de Jong |
| collection | DOAJ |
| description | Abstract Background Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. This study explored the utility of nanopore sequencing to examine whole BRCA1 mRNA transcripts and to provide accurate categorisation of in-frame and out-of-frame splicing events. Methods The exon structure of BRCA1 transcripts from a previously studied control lymphoblastoid cell line were assessed using MinION nanopore sequencing of long-range reverse transcriptase-PCR amplicons. Results Our study identified and characterised 32 complete BRCA1 isoforms, including 18 novel isoforms which showed skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as Δ(9,10) and Δ21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Fourteen novel isoforms were formed entirely from a combination of previously identified alternative splice junctions, suggesting that the total number of BRCA1 isoforms might be lower than the number of splicing events reported previously. Conclusions Our results highlight complexity in BRCA1 transcript structure that has not been described previously. This finding has key implications for predicting the translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants. |
| first_indexed | 2024-12-16T23:43:24Z |
| format | Article |
| id | doaj.art-0adda2710f3a41eda609188b081e49ae |
| institution | Directory Open Access Journal |
| issn | 1465-542X |
| language | English |
| last_indexed | 2024-12-16T23:43:24Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Breast Cancer Research |
| spelling | doaj.art-0adda2710f3a41eda609188b081e49ae2022-12-21T22:11:33ZengBMCBreast Cancer Research1465-542X2017-11-011911910.1186/s13058-017-0919-1Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping eventsLucy C. de Jong0Simone Cree1Vanessa Lattimore2George A. R. Wiggins3Amanda B. Spurdle4kConFab Investigators5Allison Miller6Martin A. Kennedy7Logan C. Walker8Department of Pathology, University of OtagoDepartment of Pathology, University of OtagoDepartment of Pathology, University of OtagoDepartment of Pathology, University of OtagoGenetics and Computational Biology Division, QIMR Berghofer Medical Research InstitutekConFab, Research Department, Peter MacCallum Cancer CentreDepartment of Pathology, University of OtagoDepartment of Pathology, University of OtagoDepartment of Pathology, University of OtagoAbstract Background Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. This study explored the utility of nanopore sequencing to examine whole BRCA1 mRNA transcripts and to provide accurate categorisation of in-frame and out-of-frame splicing events. Methods The exon structure of BRCA1 transcripts from a previously studied control lymphoblastoid cell line were assessed using MinION nanopore sequencing of long-range reverse transcriptase-PCR amplicons. Results Our study identified and characterised 32 complete BRCA1 isoforms, including 18 novel isoforms which showed skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as Δ(9,10) and Δ21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Fourteen novel isoforms were formed entirely from a combination of previously identified alternative splice junctions, suggesting that the total number of BRCA1 isoforms might be lower than the number of splicing events reported previously. Conclusions Our results highlight complexity in BRCA1 transcript structure that has not been described previously. This finding has key implications for predicting the translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.http://link.springer.com/article/10.1186/s13058-017-0919-1BRCA1SplicingMinIONNanopore sequencingFull-length transcriptExon skipping |
| spellingShingle | Lucy C. de Jong Simone Cree Vanessa Lattimore George A. R. Wiggins Amanda B. Spurdle kConFab Investigators Allison Miller Martin A. Kennedy Logan C. Walker Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events Breast Cancer Research BRCA1 Splicing MinION Nanopore sequencing Full-length transcript Exon skipping |
| title | Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events |
| title_full | Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events |
| title_fullStr | Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events |
| title_full_unstemmed | Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events |
| title_short | Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events |
| title_sort | nanopore sequencing of full length brca1 mrna transcripts reveals co occurrence of known exon skipping events |
| topic | BRCA1 Splicing MinION Nanopore sequencing Full-length transcript Exon skipping |
| url | http://link.springer.com/article/10.1186/s13058-017-0919-1 |
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