Structural and thermodynamic analysis of BRCA1 BRCT variants identified in Middle Eastern breast cancer cohorts

BRCA1 is a critical tumor suppressor that maintains genomic integrity through its C-terminal BRCT tandem domain, which mediates phosphoprotein interactions involved in DNA damage response signaling. Missense variants within the BRCT domain may disrupt protein stability and phosphopeptide recognition; however, their functional interpretation is often complicated by conflicting computational predictions and limited experimental evidence. We characterized two Middle Eastern BRCA1 BRCT variants, M1652I and F1662S, identified in Bahraini and Lebanese cohorts, respectively and compared their structural and functional properties with the pathogenic R1699W variant.

The structural stability and binding thermodynamics of wild-type and mutant BRCA1 BRCT domains were evaluated using circular dichroism, thermal denaturation assays and isothermal titration calorimetry (ITC) with phosphorylated BACH1 and CtIP peptides.

M1652I and F1662S retained secondary structure and thermal stability comparable to wild-type BRCA1, whereas R1699W exhibited partial destabilization and a reduced melting temperature (ΔT_m = -5.3 °C). ITC demonstrated that M1652I and F1662S preserved near wild-type binding affinities for phosphorylated BACH1 and CtIP peptides, while R1699W showed no detectable phosphopeptide binding under the conditions tested.

M1652I and F1662S preserve BRCT structural integrity and phosphopeptide recognition, supporting a likely benign functional effect on BRCA1 BRCT-mediated interactions. These findings highlight the value of experimental biophysical characterization for interpreting BRCA1 variants identified in underrepresented populations. A major limitation is the reliance on in vitro biophysical assays; therefore, additional cellular and in vivo functional studies are required to confirm the biological consequences of these variants.