がんの科学と治療

Pathogenicity of Mutations Discovered in BRCA1 BRCT Domains is Characterized by Destabilizing the Hydrophobic Interactions

Abstract

Dilip C Badgujar, Ulka Sawant, Hafiza Mahadik, Nikhil Gadewal and Ashok K Varma

Background: Breast and ovarian cancer are the most common cancers among hetrogenetically diversified women. It is quite difficult to categorize the population at a high risk of breast cancer using peer genetic information because one particular mutation can be found in the same or in different families. Several mutations have been discovered across the full length of BRCA1 gene, and categorizing their pathogenicity is a major challenge. Carriers of BRCA1 mutations have an increased risk of developing cancer. In the breast cancer database BIC, approximately 1,500 genetic variants have been reported. It is very difficult to characterize each of the reported mutations. Given the complexities in characterizing the mutations, we decided to investigate functional basis associated with the mutations, rather than looking at each mutation.

Materials and methods: BRCA1 BRCT domains were cloned, expressed, and purified using e-coli bacterial expression system. Mutations were generated using site-directed mutagenesis techniques, and all the mutations were sequence verified. The secondary structure of the mutant was characterized by Circular dichroism (CD) and Fluorescence spectroscopy. Molecular dynamics simulations were performed using Desmond software. Hydrophobic interactions and hydrogen bonding of docked molecules were compared using the LigPlot program.

Results: Genetic mutations were discovered throughout BRCA1, and most of the pathogenic mutations were buried in the hydrophobic core and destabilized the BRCA1 BRCT domain. This unstable BRCT domain destabilized the full-length BRCA1, resulting in a loss of function. We conclude that the pathogenicity of each of the mutations in the BRCT domain can be categorized on the basis of its ability to destabilize the hydrophobic interactions. Although such instability is not sufficient to predispose someone to cancer, it provides a basis for formulating a concept for genetic counseling and targeted therapy.