Genetic, functional and histopathological evaluation of two C-terminal BRCA1 missense variants

Paul K Lovelock ¹, Sue Healey ¹, Wendy Au ², Eleanor Sum ³, Andrea Tesoriero ⁴, Ee Ming Wong ⁴, Shannon Hinson ⁵, Ross Brinkworth ⁶, Anna Bekessy ⁷, Orland Diez ⁸, Louise Izatt ⁹, Ellen Solomon ⁹, Mark Jenkins ⁴, Helene Renard ¹⁰, John Hopper ⁴, Paul Waring ¹¹, kConFab Investigators ¹¹, Sean V Tavtigian ¹⁰, David Goldgar ¹⁰, Geoffrey J Lindeman ³, Jane E Visvader ³, Fergus J Couch ⁵, Beric R Henderson ², Melissa Southey ¹⁰, Georgia Chenevix-Trench ^7*, Amanda B Spurdle ⁷ and Melissa A Brown ⁶

¹ Queensland Institute of Medical Research, Australia
² Westmead Institute for Cancer Research, University of Sydney, Sydney, Australia
³ The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
⁴ University of Melbourne, Melbourne, Australia
⁵ Mayo Clinic College of Medicine, Rochester, Minnesota, United States
⁶ School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia
⁷ Queensland Institute of Medical Research, Brisbane, Australia
⁸ Servei de Genetica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
⁹ Division of Genetics and Molecular Medicine, King's College, Guy's Hospital, London, United Kingdom
¹⁰ International Agency for Research on Cancer, Lyon, France
¹¹ Peter MacCallum Cancer Centre, Melbourne, Australia

^* To whom correspondence should be addressed. E-mail: georgia.trench{at}qimr.edu.au.

Accepted 20 May 2005

Abstract

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene.

Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes.

Methods and Results: Data from a range of bioinformatic, genetic and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast the 1706A variant displayed a phenotype comparable to wild-type BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1-related tumours.

Conclusions: A comprehensive range of genetic, bioinformatic and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as 'benign'. In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Keywords: BRCA1, functional analysis, unclassified variants

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