Missense variants of uncertain significance (VUS) altering the phosphorylation patterns of BRCA1 and BRCA2

Abstract

Mutations in BRCA1 and BRCA2 are responsible for a large proportion of breast-ovarian cancer families. Protein-truncating mutations have been effectively used in the clinical management of familial breast cancer due to their deleterious impact on protein function. However, the majority of missense variants identified throughout the genes continue to pose an obstacle for predictive informative testing due to low frequency and lack of information on how they affect BRCA1/2 function. Phosphorylation of BRCA1 and BRCA2 play an important role in their function as regulators of DNA repair, transcription and cell cycle in response to DNA damage but whether missense variants of uncertain significance (VUS) are able to disrupt this important process is not known. Here we employed a novel approach using NetworKIN which predicts in vivo kinase-substrate relationship, and evolutionary conservation algorithms SIFT, PolyPhen and Align-GVGD. We evaluated whether 191 BRCA1 and 43 BRCA2 VUS from the Breast Cancer Information Core (BIC) database can functionally alter the consensus phosphorylation motifs and abolish kinase recognition and binding to sites known to be phosphorylated in vivo. Our results show that 13.09% (25/191) BRCA1 and 13.95% (6/43) BRCA2 VUS altered the phosphorylation of BRCA1 and BRCA2. We highlight six BRCA1 (K309T, S632N, S1143F, Q1144H, Q1281P, S1542C) and three BRCA2 (S196I, T207A, P3292L) VUS as potentially clinically significant. These occurred rarely (n<2 in BIC), mutated evolutionarily conserved residues and abolished kinase binding to motifs established in the literature involved in DNA repair, cell cycle regulation, transcription or response to DNA damage. Additionally in vivo phosphorylation sites identified via through-put methods are also affected by VUS and are attractive targets for studying their biological and functional significance. We propose that rare VUS affecting phosphorylation may be a novel and important mechanism for which BRCA1 and BRCA2 functions are disrupted in breast cancer.

Figure 1

a. Summary of phosphorylation sites studied in BRCA1. Residues in green represent in vivo phosphorylation sites have been biologically characterized in the literature. Residues in red represent in vivo phosphorylation sites identified via throughput methods where biological functions have not yet been determined. b. Summary of phosphorylation sites studied in BRCA2. Residues in green represent in vivo phosphorylation sites that have been biologically characterized in the literature. Residues in red represent in vivo phosphorylation sites identified via throughput methods where biological functions have not yet been determined.

Figure 2. Multiple sequence alignment demonstrating evolutionary conservation of the six biologically characterized phosphorylated BRCA1 residues affected by missense variants of unknown clinical significance.

Figure 3. Multiple sequence alignment demonstrating phylogenetic conservation of the three biologically characterized phosphorylated BRCA2 residues affected by missense variants of unknown clinical significance.