Inter-BRCT linker is probably the most intolerant region of the BRCA1 BRCT domain

Pathogenic mutations in BRCA1 are associated with an increased risk of hereditary breast, ovarian, and some other cancers; however, the clinical significance of many mutations in this gene remains unknown (Variants of Unknown Significance/VUS). Since mutations in intolerant regions of a protein lead to dysfunction and pathogenicity, identifying these regions helps to predict the clinical importance of VUSs. This study aimed to identify intolerant regions of BRCA1 and understand the possible root of this susceptibility. Intolerant regions appear to carry more pathogenic mutations than expected due to their lower tolerance to missense variations. Therefore, we hypothesized that among the BRCA1 regions, the higher the mutation density, the greater the intolerance. Thus, pathogenic mutation density and regional intolerance scores were calculated to identify BRCA1-intolerant regions. To investigate the pathogenic mechanisms of missense-intolerant regions in BRCA1, transcription activation (TA) experiments and molecular dynamics (MD) simulations were also performed. The results showed that the RING domain, followed by the BRCT domain, has the highest density of pathogenic mutations. In the BRCT domain, a higher density of pathogenic mutations was observed in the inter-BRCT linker. Additionally, scores generated by Missense Tolerance Ratio-3D (MTR3D) and the Missense Tolerance Ratio consensus (MTRX) showed that the inter-BRCT linker is more intolerant than other regions of the BRCT domain. The MD results showed that mutations in the inter-BRCT linker led to cancer susceptibility, likely due to disruption of the interaction between BRCA1 and phosphopeptides. TA laboratory assays further supported the importance of the inter-BRCT linker.Communicated by Ramaswamy H. Sarma.