BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression

Abstract

The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82-94%), poor for BRCAX with an LCS (40-50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71-100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.

Figure 1. Unsupervised Hierarchical cluster of differences between BRCAX samples with or without a BRCA1 or BRCA2 sequence variant of Low Clinical Significance.

Unsupervised cluster analysis was performed using 1778 genes that varied in expression 2-fold from the mean in 10% of BRCAX without a BRCA1/2 LCS variant and BRCAX samples with an LCS. The tree structure at the top of the cluster shows how related the samples are to each other. The majority of BRCAX samples without an LCS (black) clustered in a distinct group away from BRCAX with an LCS variant (red).

Figure 2. Unsupervised Hierarchical Cluster of Differences between BRCA1, BRCA2 and BRCAX samples.

Clustering was based on 4751 genes which varied 2-fold difference in gene expression in at least 10% of samples. There are two main clusters, the BRCAX samples without a BRCA1/2 LCS variant (black) cluster to the right, whereas BRCA1 (green), BRCA2 (blue) and BRCAX samples with a BRCA1/2 LCS variant (red) are predominantly located in the left cluster. The missense pathogenic mutations of BRCA1 or BRCA2 are indicated with arrows and 6/9 cluster closest to the BRCAX LCLs.

Figure 3. Venn Diagrams of Differences between BRCA1 or BRCA2 and BRCAX LCLs with or without a LCS.

T-tests (p-value<0.05) were performed to determine genes that differed between LCLs as follows: A) BRCA1 Truncating mutations vs BRCAX with no LCS, and BRCA1 missense mutations vs BRCAX with no LCS; B) BRCA1 Truncating mutations vs BRCAX with an LCS, and BRCA1 missense mutations vs BRCAX with an LCS. C) BRCA2 Truncating mutations vs BRCAX with no LCS, and BRCA2 missense mutations vs BRCAX with no LCS; D) BRCA2 Truncating mutations vs BRCAX with an LCS, and BRCA1 missense mutations vs BRCAX with an LCS. For each comparison, the overlap of genes is shown.

Figure 4. Confidence of Predictions for Missense BRCA1 LCLs as Determined by Distance from the SVM Plane.

The SVM plane separating BRCA1 from BRCAX is shown by the red line. If the sample falls over the line (black point) the missense mutation is correctly predicted as pathogenic for BRCA1 mutation. If the sample falls under the line (red point) the missense mutation is incorrectly predicted as BRCAX. The gene lists used for the predictions are the top 200 genes from BRCA1 missense vs BRCAX, and the top 200 genes from BRCA1 Truncating vs BRCAX.