Abstract
A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.
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Acknowledgements
We thank the staff at the NSLS for assistance during data collection and Dr Donald Coen at Harvard Medical School for providing access to the microcalorimeter facility. This study was supported by grants DK062162 and AG021964 from the National Institutes of Health, and DAMD170210300 and DAMD170310563 from the US Department of Defense to JAAL, by the Jewish General Hospital Weekend to End Breast Cancer, Rethink Breast Cancer Canada, The Canadian Foundation for Innovation (MT), the Canadian Breast Cancer Research Alliance (WDF), Florida Breast Cancer Coalition, and National Institutes of Health CA92309 and CA116167 (ANAM) and the Dutch Cancer Society/Koningin Wilhelmina Fonds (PMN). We thank Heather Thorne, Sue Healey (QIMR), Eveline Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (funded by NHMRC grants 145684 and 288704 and 454508) for their contributions to this resource, and the many families who contribute to kConFab. kConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. WDF holds a Fonds de la Recherche en Santé du Québec (FRSQ) national scientist award and MT holds a FRSQ clinician-scientist award.
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Tischkowitz, M., Hamel, N., Carvalho, M. et al. Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach. Eur J Hum Genet 16, 820–832 (2008). https://doi.org/10.1038/ejhg.2008.13
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DOI: https://doi.org/10.1038/ejhg.2008.13
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