Functional assessment of protein variants in structured domains by fluorescence cross-correlation spectroscopy

With the expanding catalogue of novel disease-genes, there is an increasing need to establish the significance of potential disease-causing variants. Based on the idea that pathogenic variants in structured protein domains disturb folding and association with macromolecular assemblies, we employed Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS) to assess in vivo protein complex formation. Since the molecular underpinning of BRCA-associated breast and ovarian cancers is well defined and data from a recent genome editing screening allowed us to compare variant binding data with a reliable functional HRD test in addition to ClinVar and AlphaMissense data, we examined the binding of mutated full-length BRCA1 or isolated RING and BRCT domains to BARD1 and RBBP8, respectively. The results demonstrate that FCCS, whether applied to full-length BRCA1 in live cells and/or to isolated domains in cellular lysates, identified pathogenic BRCA1 RING or BRCT domain variants. We moreover demonstrate the feasibility of employing FCCS for analysis of HNPCC-related factor MSH2 and MEN1 factor Menin variants in combination with DNA mismatch repair factor MSH6 and transcription factor JUND, respectively. We propose that FCCS may be an appealing complement to current clinical procedures for classifying variants for many monogenic diseases, given its generic nature and ease of use.