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RNA analysis of cancer predisposing genes in formalin-fixed paraffin-embedded tissue determines aberrant splicing.
- Source :
-
European journal of human genetics : EJHG [Eur J Hum Genet] 2018 Aug; Vol. 26 (8), pp. 1143-1150. Date of Electronic Publication: 2018 Apr 30. - Publication Year :
- 2018
-
Abstract
- High-throughput sequencing efforts in molecular tumour diagnostics detect increasing numbers of novel variants, including variants predicted to affect splicing. In silico prediction tools can reliably predict the effect of variant disrupting canonical splice sites; however, experimental validation is required to confirm aberrant splicing. Here, we present RNA analysis performed for 13 canonical splice site variants predicted or known to result in splicing in the cancer predisposition genes MLH1, MSH2, MSH6, APC and BRCA1. Total nucleic acid was successfully isolated for 10 variants from eight formalin-fixed paraffin-embedded (FFPE) tumour tissues and two B-cell lines. Aberrant splicing was confirmed in all six variants known to result in splicing. Of one known variant in the B-cell line, aberrant splicing could only be detected after formalin fixation, which indicated that formalin fixation could possibly inhibit RNA degradation. Aberrant splicing was concluded in three of four predicted splice variants of uncertain significance, supporting their pathogenic effect. With this assay, somatic splice variants can be easily and rapidly analysed, enabling retrospective analysis to support the pathogenicity of variants predicted to result in splicing when only FFPE material is available.
- Subjects :
- Adenomatous Polyposis Coli Protein genetics
BRCA1 Protein genetics
Breast Neoplasms pathology
DNA-Binding Proteins genetics
Female
Genetic Testing standards
Humans
MutL Protein Homolog 1 genetics
MutS Homolog 2 Protein genetics
Sequence Analysis, RNA standards
Tissue Embedding methods
Tissue Embedding standards
Tissue Fixation methods
Tissue Fixation standards
Breast Neoplasms genetics
Genetic Testing methods
Mutation
RNA Splicing
Sequence Analysis, RNA methods
Subjects
Details
- Language :
- English
- ISSN :
- 1476-5438
- Volume :
- 26
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- European journal of human genetics : EJHG
- Publication Type :
- Academic Journal
- Accession number :
- 29706640
- Full Text :
- https://doi.org/10.1038/s41431-018-0153-z