Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants : Application of a points-based ACMG/AMP approach
(2022) In Human Mutation 43(12). p.1921-1944- Abstract
Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a... (More)
Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.
(Less)
- author
- author collaboration
- organization
- publishing date
- 2022-12
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- ACMG/AMP classification, BRCA2, dPCR, functional analysis, quantitation, splicing
- in
- Human Mutation
- volume
- 43
- issue
- 12
- pages
- 24 pages
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:35979650
- scopus:85140357391
- ISSN
- 1059-7794
- DOI
- 10.1002/humu.24449
- language
- English
- LU publication?
- yes
- id
- 171bb2ac-6d6f-4e70-b3bd-ef4481a955c7
- date added to LUP
- 2023-01-16 13:20:40
- date last changed
- 2024-06-13 01:33:10
@article{171bb2ac-6d6f-4e70-b3bd-ef4481a955c7,
abstract = {{<p>Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.</p>}},
author = {{Thomassen, Mads and Mesman, Romy L.S. and Hansen, Thomas V.O. and Menendez, Mireia and Rossing, Maria and Esteban-Sánchez, Ada and Tudini, Emma and Törngren, Therese and Parsons, Michael T. and Pedersen, Inge S. and Teo, Soo H. and Kruse, Torben A. and Møller, Pål and Borg, Åke and Jensen, Uffe B. and Christensen, Lise L. and Singer, Christian F. and Muhr, Daniela and Santamarina, Marta and Brandao, Rita and Andresen, Brage S. and Feng, Bing Jian and Canson, Daffodil and Richardson, Marcy E. and Karam, Rachid and Pesaran, Tina and LaDuca, Holly and Conner, Blair R. and Abualkheir, Nelly and Hoang, Lily and Calléja, Fabienne M.G.R. and Andrews, Lesley and James, Paul A. and Bunyan, Dave and Hamblett, Amanda and Radice, Paolo and Goldgar, David E. and Walker, Logan C. and Engel, Christoph and Claes, Kathleen B.M. and Macháčková, Eva and Baralle, Diana and Viel, Alessandra and Wappenschmidt, Barbara and Lazaro, Conxi and Vega, Ana and Vreeswijk, Maaike P.G. and de la Hoya, Miguel and Spurdle, Amanda B.}},
issn = {{1059-7794}},
keywords = {{ACMG/AMP classification; BRCA2; dPCR; functional analysis; quantitation; splicing}},
language = {{eng}},
number = {{12}},
pages = {{1921--1944}},
publisher = {{John Wiley & Sons Inc.}},
series = {{Human Mutation}},
title = {{Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants : Application of a points-based ACMG/AMP approach}},
url = {{http://dx.doi.org/10.1002/humu.24449}},
doi = {{10.1002/humu.24449}},
volume = {{43}},
year = {{2022}},
}