Resolving the Functional Significance of BRCA1 RING Domain Missense Substitutions

Development and calibration of suitably accurate functional assays for BRCA1 RING domain and BRCT domain missense substitutions could dramatically accelerate clinical classification of rare missense substitutions observed in that gene. Leveraging data from 68,000 full sequence tests of BRCA1 and BRCA2, plus data from the limited number of already classified BRCA1 RING domain missense substitutions, we used logistic regression and related techniques to evaluate three BRCA1 RING domain assays. These were recently described high throughput yeast 2-hybrid and E3 ubiquitin ligase assays, plus a newly developed mammalian 2- hybrid assay. While there were concerns about the accuracy of the yeast 2-hybrid assay and the indirect nature of the ubiquitin ligase assay, the mammalian 2-hybrid assay had excellent correlation with existing missense substitution classifications. After calibration, this assay contributed to classification of one newly reported BRCA1 missense substitution. In principal, the mammalian 2-hybrid assay could be converted to a high-throughput format that would likely retain suitable accuracy.