Rare BRIP1 Missense Alleles Confer Risk for Ovarian and Breast Cancer

Abstract

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ ² test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60-3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. SIGNIFICANCE: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Figure 1.. Graphical summary of BRIP1 rare (MAF<0.0005) and novel missense variants identified in ovarian and early-onset breast cancer patients.

The ATPase reduced, helicase deficient P47A (helicase domain I) was seen in both cohorts. Helicase deficient, dominant negative mutant A349P (Fe-S domain, 276-362aa) was identified in a single ovarian cancer patient. Rare alleles in the C-terminal region of the helicase domain (Q540L, I691L, Q740H and A745T) identified in both cohorts are indicated with black arrowheads. Mutation plot generated using cBioPortal MutationMapper tool. Helicase domain motifs: I (39-57); Ia (245-258); II (385-398); III (610-624); IV (689-710); V (748-775); VI (819-836). BRCA1 binding domain: 888-1063aa.

Figure 2.. Missense alleles in BRIP1 increase sensitivity to ICL damage.

A) Representative karyotypes of independent HeLa clones after exposure to MMC [60nM]. Chromosomal breaks, gaps, radials (red arrows) and acentric fragments (yellow arrows) were observed in −/−, A349P/-, P47A/- and D791V/−. The average number of breaks/gaps, radial formations and acentric fragments per metaphase spread were calculated for each genotype (n=15 spreads per cell line). Statistical significance determined by two way ANOVA with a Bonferroni posttest to BRIP1+/+ (*** p<0.0001). B) Overlay of representative cell cycles from independent HeLa clones with and without MMC treatment. Statistical significance calculated by averaging percentage of cells in G2/M from 3 replicates (one-way ANOVA with Tukey’s multiple comparison test).

Figure 3.. Missense alleles classified as null, hypomorphic or wildtype based on ICL damage induced cell killing.

Clonogenic growth assays of independent HeLa clones after exposure to increasing concentrations of MMC and cisplatin. Data presented are the average of three replicates with error bars indicating one standard deviation. Cisplatin experiments were performed simultaneously with a single BRIP1+/+. +/− and −/− clone for control.

Figure 4.. BRIP1 hypomorphs exhibit protein instability.

A) Representative western blot showing variable protein expression in HeLa clones (red arrowhead denotes BRIP1 protein; black arrow head denotes non-specific band of lower mass). Right panel: Quantification of relative BRIP1 levels. Data presented are the average of three replicates with error bars indicating one standard deviation. Statistical significance determined by one way ANOVA with a Dunnett’s multiple comparison test to BRIP1+/+ (*** p<0.0001). B) Cycloheximide chase analysis of endogenous wildtype and A349P protein. Fifty percent protein expression denoted by dotted black line. Data presented are the average of three replicates with error bars indicating one standard deviation. C) Cycloheximdie chase analysis of exogenously expressed wildtype, A349P, A745T, D791V and P47A protein. Half-life denoted by dotted black line. Data presented are the average of three replicates with error bars indicating one standard deviation.

Figure 5.. Occurrence of functionally characterized variants in clinical testing.

Q740H and A745T account for a majority of the wildtype alleles (green) identified. P47A was the most common hypomorph allele (orange) detected. Null alleles (red) were seen at lower frequencies. Helicase domain motifs: III (610-624); IV (689-710); V (748-775); VI (819-836).