Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach
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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.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Human Mutation |
| Vol/bind | 43 |
| Udgave nummer | 12 |
| Sider (fra-til) | 1921-1944 |
| ISSN | 1059-7794 |
| DOI | |
| Status | Udgivet - 2022 |
Bibliografisk note
Funding Information:
The authors thank J. Jonkers and P. Bouwman (Netherlands Cancer Institute, Amsterdam, the Netherlands) for the I‐Sce1‐mCherry plasmid; S.K. Sharan (National Cancer Institute at Frederick, Frederick, USA) for the Pl2F7 conditional Brca2 knockout mES cell line (PMID: 18607349); M. Jasin (Memorial Sloan‐Kettering Cancer Center, New York, USA) for the DR‐GFP reporter plasmid (PMID: 11239455). The authors wish also to thank all the members of the ICO Hereditary Cancer Program. LCW was supported by the Royal Society of New Zealand Rutherford Discovery Fellowship. The FPGMX thanks members of the Cancer Genetics group (IDIS): Miguel Aguado, Olivia Fuentes, and Ana Crujeiras. The work of MPGV was financially supported by the Dutch Cancer Society KWF (UL2012‐5649 and Pink Ribbon‐11704). The work by PM was supported by a “Pink Ribbon” grant #194751 from Den Norske Kreftforening to E.H. The work of MdlH was supported by Spanish Instituto de Salud Carlos III (ISCIII) funding grant PI 20/00110, an initiative of the Spanish Ministry of Economy and Innovation. ABS, MTP and ET were supported by NHMRC Funding (APP177524, APP1104808). The work by CL and MM received institutional support by CERCA Program/Generalitat de Catalunya and grant support by the Carlos III National Health Institute funded by FEDER funds—a way to build Europe—[PI19/00553; PI16/00563; SAF2015‐68016‐R and CIBERONC]; the Government of Catalonia [Pla estratègic de recerca i innovació en salut (PERIS_MedPerCan and URDCat projects), 2017SGR1282 and 2017SGR496]. The Baralle lab is supported by NIHR Research Professorship to DB (RP‐2016‐07‐011). The work of AV was supported by the Spanish Health Research Foundation, Instituto de Salud Carlos III (ISCIII) through Research Activity Intensification Program (contract grant numbers: INT15/00070, INT16/00154, INT17/00133, and INT20/00071), and through Centro de Investigación Biomédica en Red de Enfermedades Raras CIBERER (ACCI 2016: ER17P1AC7112/2018); Autonomous Government of Galicia (Consolidation and structuring program: IN607B), and by the Fundación Mutua Madrileña (call 2018). The German Consortium of Hereditary Breast and Ovarian Cancer (GC‐HBOC) is supported by the German Cancer Aid (Grant nos. 110837 and 70114178), (RKS) and by the Federal Ministry of Education and Research (Grant no. 01GY1901), (RKS). The work of EMA was supported by the Ministry of Health of the Czech Republic MH CZ—DRO (MMCI, 00209805) and AZV project NU20‐03‐00285. Institutional support by Italian Ministy of Health, Ricerca Corrente of CRO Aviano, Line 1 (AVI).
Publisher Copyright:
© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.
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