A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers

Abstract

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Figure 1. The genomic landscape of somatic alterations in BRCA1 ER-negative and ER-positive breast cancers.

Circos plots of (A) BRCA1 mutant ER-negative (BRCA1/ER-BC) and (B) BRCA1 mutant ER-positive (BRCA1/ER+BC) primary breast cancers, derived from massively parallel sequencing data at a coverage of >30x of the genome (Supplementary Table S1). Circles from outside to inside depict the following: i) chromosomes and validated mutations above the somatic score threshold (Supplementary Methods S1); ii) copy number derived from GC normalised sequence coverage (2kb window). Red indicates copy number loss, green copy number gain and bright green amplifications; iii) high-confidence structural rearrangements (grey), with rearrangements validated by Sanger sequencing highlighted in purple.

Figure 2. Mutation rates in BRCA1 mutant ER-negative and ER-positive breast cancers.

Mutation rates as defined by single nucleotide variants (SNVs) per mega-base pair (Mbp), above the somatic score threshold (i.e. BRCA1/ER-BC-0.057 and BRCA1/ER+BC-0.034), in BRCA1/ER-BC (dark blue) and BRCA1/ER+BC (light blue) for whole-genome (WG), genic regions, intergenic regions, exonic regions, intronic regions, expressed genes, and non-expressed genes. P values: two-tailed Fisher’s exact test. SNV: single nucleotide variation.

Figure 3. Mutational trends and patterns in BRCA1 mutant ER-negative and ER-positive breast cancers.

Frequency of transitions to transversions in BRCA1/ER-BC (dark blue) and BRCA1/ER+BC (light blue) in the germline (A) and tumour (B) DNA samples of BRCA1/ER-BC (dark blue) and BRCA1/ER+BC (light blue). The y-axis depicts the frequency of base changes. In both tumours, the ratio of transition:transversion decreases in comparison to the germline ratio of 2:1. The sequence context of somatic substitutions of C>G/G>C transversions in BRCA1/ER-BC (C) and in BRCA1/ER+BC (D). The bars at position zero indicate the variation itself. The remaining bars indicate the fractions of different nucleotides at positions 5’ (minus) and 3’ (plus) to the variation. C>G/G>C transversions were more frequently preceded and/or succeeded by T. Chi-square logarithm (base 10) of odds (LOD) scores are depicted below each panel. Chi-square LOD scores of > 2 were considered significant.

Figure 4. Landscape of structural rearrangements in BRCA1 breast cancers.

Summary of the landscape of high-confidence structural rearrangements in BRCA1/ER-BC (dark blue) and BRCA1/ER+BC (light blue) according to individual classes of structural rearrangements (A). Total number of structural rearrangements in BRCA1/ER-BC (dark blue) and BRCA1/ER+BC (light blue) that are intra-chromosomal and inter-chromosomal (B). Number of validated structural variants predicted to be in-frame and not in-frame (C). Number of structural variants validated by Sanger sequencing that were expressed and not expressed at the RNA level (D). The number of structural rearrangements is plotted in the y-axis. P values: two-tailed Fisher’s exact test. Extent of overlapping micro-homology sequences at structural rearrangement breakpoints in BRCA1/ER-BC (E) and in BRCA1/ER+BC (F). The number of structural rearrangements (y-axis) is plotted according to the number of base pairs of micro-homology (x-axis).