Use of BRCA1 protein:protein interactions to classify cancer risk

Germline loss-of-function mutations in BRCA1 are associated with a high lifetime risk of breast and ovarian cancer. Most mutations in the gene are 'truncating': in the main these induce premature termination codons, resulting in nonsense-mediated decay, loss of the transcript and/or the entire protein. The improved screening methods now in use across the UK will identify many carriers of unclassified BRCA1 variants. These are chiefly missense mutations, introducing an amino acid change in the context of an expressed protein. Indeed more than one-quarter of entries recorded in the Breast Cancer Information Core dataset of BRCA1 sequence variants collected from patients worldwide are unclassified missense alterations http://research.nhgri.nih.gov/bic/. Currently, discovery of the majority of missense variants leaves both variant carriers and their families in an ambiguous position.

These variants remain unclassified because in the majority of cases it is not possible to follow variants by cosegregation analysis, and the number of appropriate controls required to be certain that a variant is absent in unaffected individuals is prohibitive. Currently, in silico algorithms try to distinguish between missense substitutions that are likely to be pathogenic and those that are not. These algorithms compile a multicomponent likelihood ratio that integrates assessment methods ranging from conservation analysis, co-occurrence of a deleterious allele in trans, and immuno-histochemical analysis [1–3]. What is missing from these analyses is the relationship between loss of protein function and detriment to patient health.

We have focused on the N-terminal region of BRCA1. This region has a high density of missense substitutions, including those of known pathogenic status, and many currently unclassified variants. We have shown that experimental missense variants, generated randomly and selected for loss of interaction with the BRCA1 ubiquitin ligase components, BARD1 and the E2 enzyme UbcH5, identify variants reported within the Breast Cancer Information Core database of individuals with a personal or family history of breast cancer [4]. The E2 component is particularly sensitive to missense alteration in BRCA1, with the majority of currently unclassified variants in the region inhibiting interaction, whereas the BARD1 component is disrupted by a smaller, but overlapping, subset restricted to substitution of the structurally detrimental zinc-ligation residues. Variants that inhibited the E2 also prevented the enzymatic activity. These data strongly suggest that the ligase activity of BRCA1, through interaction with E2 and BARD1, is related to breast cancer predisposition.

Using yeast two-hybrid analysis for BRCA1:BARD1 and BRCA1:E2 interaction, we have tested the most chemically different substitutions achievable by single nucleotide change in all of the most highly conserved amino acids of the region (invariant from human to sea urchin), and have also tested all currently identified patient missense variants. These data have been combined with Grantham variation and Grantham deviance scores (a measure of how conserved an amino acid is, together with how different the protein change is) to assess the relationship between protein:protein interaction and measures of disease risk. Risk measures were based on the results of full sequence tests of BRCA1 and BRCA2 from 68,000 BRAC Analysis subjects (Myriad Genetics, Salt Lake City, UT, USA), and used estimates of the odds of developing breast cancer for a carrier of a BRCA1 missense substitution [2], together with enrichment ratios achieved by comparing the variants observed in the dataset with the variants expected on the basis of known substitution rates.

Classification methods in the past have attempted to place variants in either the pathogenic or the little-clinical significance categories. The results of this analysis suggest that some classes of variant may confer an intermediate risk. If so, these data have considerable implications for the counselling and clinical management of women found to be positive for missense variants in future.