Missense Variants of Uncertain Significance (VUS) Altering the Phosphorylation Patterns of BRCA1 and BRCA2

Eric Tram,Hilmi Ozcelik,Sevtap Savas,Amanda Ewart Toland

doi:10.1371/journal.pone.0062468

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.

Highlights

Rare germline mutations of BRCA1 and BRCA2 predispose carriers to early-onset familial breast or ovarian cancers [1,2,3]
Our studies have previously suggested that missense variants of uncertain significance (VUS) and commonly occurring single nucleotide polymorphisms (SNPs) altering phosphorylation patterns of cell cycle and DNA repair proteins may contribute to human cancer risk [24,25] and our preliminary analysis showed that many of the missense variants in Breast Cancer Information Core (BIC) are found within the consensus motifs of sites known to be phosphorylated in vivo
Our analysis indicated that 13.09% (25/191) BRCA1 and 13.95% (6/43) BRCA2 VUSs impact an existing phosphorylation site, and/or create a new site at the altered residue (Table 1, 2)

Summary

Introduction

Rare germline mutations of BRCA1 and BRCA2 predispose carriers to early-onset familial breast or ovarian cancers [1,2,3]. These include integrating interspecies sequence variation [8,9,10], functional analysis to uncover the consequences of VUS on protein function [11,12,13,14], genetic assessment approaches including pedigree analysis [15], likelihood models [16], structural-based approaches to model the effect of amino acid substitution [17,18] and transcriptional activity assays [19]. These studies have provided important information into the clinical significance of BRCA mutations

Objectives

Methods

Results

Discussion

Conclusion