Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

Meryem Ozgencil; Julian Barwell; Marc Tischkowitz; Louise Izatt; Ian Kesterton; Michael Simpson; Paul Sharpe; Paulo de Sepulveda; Edwige Voisset Ellen Solomon

doi:10.1371/journal.pone.0260852

Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

Meryem Ozgencil, Julian Barwell, Marc Tischkowitz, Louise Izatt, Ian Kesterton, Michael Simpson, Paul Sharpe, Paulo de Sepulveda^*, Edwige Voisset Ellen Solomon

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

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Abstract

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a H2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Original language	English
Article number	e0260852
Journal	PLoS ONE
Volume	16
Issue number	12
DOIs	https://doi.org/10.1371/journal.pone.0260852
Publication status	Published - Dec 2021

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Ozgencil, M., Barwell, J., Tischkowitz, M., Izatt, L., Kesterton, I., Simpson, M., Sharpe, P., de Sepulveda, P., & Solomon, E. V. E. (2021). Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance. PLoS ONE, 16(12), Article e0260852. https://doi.org/10.1371/journal.pone.0260852

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title = "Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance",

abstract = "Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a H2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.",

author = "Meryem Ozgencil and Julian Barwell and Marc Tischkowitz and Louise Izatt and Ian Kesterton and Michael Simpson and Paul Sharpe and {de Sepulveda}, Paulo and Solomon, {Edwige Voisset Ellen}",

note = "Publisher Copyright: {\textcopyright} 2021 Ozgencil et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

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month = dec,

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Ozgencil, M, Barwell, J, Tischkowitz, M, Izatt, L, Kesterton, I, Simpson, M , Sharpe, P, de Sepulveda, P & Solomon, EVE 2021, 'Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance', PLoS ONE, vol. 16, no. 12, e0260852. https://doi.org/10.1371/journal.pone.0260852

TY - JOUR

T1 - Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

AU - Ozgencil, Meryem

AU - Barwell, Julian

AU - Tischkowitz, Marc

AU - Izatt, Louise

AU - Kesterton, Ian

AU - Simpson, Michael

AU - Sharpe, Paul

AU - de Sepulveda, Paulo

AU - Solomon, Edwige Voisset Ellen

N1 - Publisher Copyright: © 2021 Ozgencil et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2021/12

Y1 - 2021/12

N2 - Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a H2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

AB - Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a H2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

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ER -

Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

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