Discussion
Although we have presented results of analyses examining risk, our goal was not to estimate penetrance per se, but rather to compare these two specific variants with the penetrance of the 'average' BRCA1 mutation (the vast majority of which are truncating), both in terms of family histories of probands carrying these variants and in terms of cosegregation of the variants within families. Here we provide, for the first time, significant evidence that a BRCA1 variant can be associated with reduced risks of breast cancer compared with the 'average' pathogenic mutation. It is of particular relevance and consequence for future studies, since the variant R1699Q was selected for study due to its behaviour in a variety of functional assays. Depending on the assay, this missense variant has demonstrated either wild-type function, abrogated function akin to known pathogenic mutations, or functional activity intermediate between that observed for wild-type BRCA1 and known truncating pathogenic and missense pathogenic mutations.
Interestingly, there was also evidence that the R1699W variant was associated with significantly lower breast cancer risk and a markedly increased risk of ovarian cancer. We recognise that the estimation of breast and ovarian cancer parameters, separately, is somewhat difficult given the necessity of conditioning the data on all pedigree phenotypes, but the results, nevertheless, raise the question that differences in risk of breast versus ovarian cancer may be a characteristic of some missense mutations in the BRCT repeat domains. In this regard, we note that in the 34 R1699W families, there were an average of 2.24 breast cancers and 1.48 ovarian cancers, while in the 68 R1699Q families, there were 2.35 breast cancers and 0.85 ovarian cancers per family, consistent with the higher estimated risk of ovarian cancer in these families. Further study of a large number of such variants will be necessary to address such an intriguing possibility that would have clear clinical implications.
Using the standard multifactorial model, the posterior probability for R1699Q is calculated to be 0.79 from the available data, namely: prior probability of pathogenicity of 0.66 based on the A-GVGD class C35; segregation odds of 5:1 in favour of pathogenicity from this study of 30 families; LRs from Easton et al of 8:1 against pathogenicity for family history, and 3:1 in favour of pathogenicity for co-occurrence data. That is, using the model developed based on the characteristics of BRCA1 pathogenic mutations of 'average' penetrance, R1699Q would be classified as International Agency for Research on Cancer (IARC) Class 3 'uncertain'.
Our conclusive finding that BRCA1 c.5096G>A R1699Q can be shown to have both intermediate functional deficiency in several assays, and is associated with breast and ovarian cancer risk at significantly lower levels than truncating BRCA1 mutations, has a number of consequences. Our findings suggest that results from a battery of functional assays may highlight other variants with intermediate or equivocal results for investigation as potential moderate risk variants. Indeed, the variant BRCA1 A1708V showed abrogated centrosome amplification, but normal nuclear foci formation and trypsin sensitivity equivocal results from a series of functional assays in our original report, and is a candidate for further investigation as a potential moderate risk variant.
If this observation of intermediate function translating to intermediate risk is a general finding, it is likely that there will be a subset of variants that are difficult to classify using the standard multifactorial likelihood approaches that are based on comparing data for a particular variant under the hypothesis that it is a fully penetrant pathogenic BRCA1 mutation, against the hypothesis that it is neutral, or of no clinical significance, with respect to risk. As shown for the R1699Q variant with more families available for analysis than will likely be achieved for most other rare variants, the standard cosegregation analysis yielded odds of only 5:1 in favour of the variant being pathogenic compared with the >6000:1 odds when a lower penetrance was allowed. Further, and more importantly, we must now face the question of how these women should be counselled in terms of cancer risk and the management of that risk. We do not propose that counselling be any different for R1699W, although results from the two parameter analyses suggest that particular attention should perhaps be paid to ovarian cancer for this known pathogenic variant. We emphasise, however, that the CI are wide, particularly for cancer site-specific risks, and future studies are necessary to confirm the markedly increased ovarian cancer risk observed in our dataset. While there is certainly significant evidence that R1699Q carriers are at increased risk over population rates, this risk is markedly lower than that observed for the average BRCA1 mutation. The findings presented here are likely to provide impetus for research studies considering approaches to clinical management of patients with cancer risks intermediate to those conferred by BRCA1/2 mutations, and those from family history alone. In the case of R1699Q, counselling could be similar to that for other moderate-penetrance genes such as PALB2, CHEK2 and RAD51C, although that may change if ovarian cancer screening improves given the increased rate of ovarian cancer over the general population. In all these cases, the incorporation of the now 30+ common breast cancer susceptibility alleles into comprehensive risk prediction models will be of great value in allowing women and their providers to make informed management decisions. In addition, it would be interesting to specifically explore if BRCA1 haplotypes altering promoter activity, or potentially altering 3' untranslated region (UTR) microRNA binding, influence the level of function of R1699Q in vivo, and explain in part the variable presentation of families.
In summary, we provide evidence that a BRCA1 variant demonstrating equivocal functional deficiency across multiple assays is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.
Appendix A