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 clinical 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 the binding data with a reliable functional HRD test, we examined the binding of BRCA1 to BARD1 and RBBP8, respectively. The results demonstrate that FCCS, whether applied to full-length BRCA1 in live cells or to isolated domains in cellular lysates, reliably identified pathogenic BRCA1 RING or BRCT 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. Because the procedure can be completed within a clinically relevant time frame, FCCS is an appealing complement to current clinical procedures for classifying variants. Given its generic nature and design, the approach can be applied to a variety of monogenic diseases. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: https://www.ncbi.nlm.nih.gov/clinvar/ I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors