Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

Abstract Background Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants,...

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Autores principales:	Amandine Billaud, Louise-Marie Chevalier, Paule Augereau, Jean-Sebastien Frenel, Christophe Passot, Mario Campone, Alain Morel
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Variants of uncertain significance Genome editing Functional testing BRCA1 BRCA2 POLE Medicine R Genetics QH426-470
Acceso en línea:	https://doaj.org/article/79c1e3cddf6545f2a4bd74aab526e620
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spelling	oai:doaj.org-article:79c1e3cddf6545f2a4bd74aab526e6202021-11-14T12:27:35ZFunctional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes10.1186/s13073-021-00976-x1756-994Xhttps://doaj.org/article/79c1e3cddf6545f2a4bd74aab526e6202021-11-01T00:00:00Zhttps://doi.org/10.1186/s13073-021-00976-xhttps://doaj.org/toc/1756-994XAbstract Background Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients’ access to targeted therapies. Methods We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS. Results For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients’ responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal. Conclusions Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients’ responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.Amandine BillaudLouise-Marie ChevalierPaule AugereauJean-Sebastien FrenelChristophe PassotMario CamponeAlain MorelBMCarticleVariants of uncertain significanceGenome editingFunctional testingBRCA1BRCA2POLEMedicineRGeneticsQH426-470ENGenome Medicine, Vol 13, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Variants of uncertain significance Genome editing Functional testing BRCA1 BRCA2 POLE Medicine R Genetics QH426-470
spellingShingle	Variants of uncertain significance Genome editing Functional testing BRCA1 BRCA2 POLE Medicine R Genetics QH426-470 Amandine Billaud Louise-Marie Chevalier Paule Augereau Jean-Sebastien Frenel Christophe Passot Mario Campone Alain Morel Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
description	Abstract Background Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients’ access to targeted therapies. Methods We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS. Results For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients’ responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal. Conclusions Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients’ responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.
format	article
author	Amandine Billaud Louise-Marie Chevalier Paule Augereau Jean-Sebastien Frenel Christophe Passot Mario Campone Alain Morel
author_facet	Amandine Billaud Louise-Marie Chevalier Paule Augereau Jean-Sebastien Frenel Christophe Passot Mario Campone Alain Morel
author_sort	Amandine Billaud
title	Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
title_short	Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
title_full	Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
title_fullStr	Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
title_full_unstemmed	Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
title_sort	functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes
publisher	BMC
publishDate	2021
url	https://doaj.org/article/79c1e3cddf6545f2a4bd74aab526e620
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Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes

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