Exome Sequencing in a Family with Luminal-Type Breast Cancer Underpinned by Variation in the Methylation Pathway

Abstract

Panel-based next generation sequencing (NGS) is currently preferred over whole exome sequencing (WES) for diagnosis of familial breast cancer, due to interpretation challenges caused by variants of uncertain clinical significance (VUS). There is also no consensus on the selection criteria for WES. In this study, a pathology-supported genetic testing (PSGT) approach was used to select two BRCA1/2 mutation-negative breast cancer patients from the same family for WES. Homozygosity for the MTHFR 677 C>T mutation detected during this PSGT pre-screen step was considered insufficient to cause bilateral breast cancer in the index case and her daughter diagnosed with early-onset breast cancer (<30 years). Extended genetic testing using WES identified the RAD50 R385C missense mutation in both cases. This rare variant with a minor allele frequency (MAF) of <0.001 was classified as a VUS after exclusion in an affected cousin and extended genotyping in 164 unrelated breast cancer patients and 160 controls. Detection of functional polymorphisms (MAF > 5%) in the folate pathway in all three affected family members is consistent with inheritance of the luminal-type breast cancer in the family. PSGT assisted with the decision to pursue extended genetic testing and facilitated clinical interpretation of WES aimed at reduction of recurrence risk.

Keywords: breast cancer; exome sequencing; methylation pathway; pathology; pharmacogenomics.

Figure 1

Outline of the pathology-supported genetic testing (PSGT) strategy used to select the index case with bilateral BRCA1/2 mutation-negative breast cancer for whole exome sequencing (WES). Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status was determined using immunohistochemistry (IHC). These features, including tumour size, grade and nodal status were evaluated to determine eligibility for MammaPrint. Based on the low-risk recurrence score obtained with this microarray test, tamoxifen treatment was administered without addition of chemotherapy. Termination of tamoxifen after one year due to adverse drug response led to pharmacogenetic CYP2D6 genotyping, which was performed as part of a chronic disease screen incorporating the cardiovascular disease (CVD) multi-gene assay. Detection of the methylenetetrahydrofolate reductase (MTHFR) 677 C>T mutation identified dysfunction of the folate pathway as a potential target for recurrence risk reduction in this overweight patient. Evaluation of family history, clinical characteristics, and laboratory test results at the protein (IHC), RNA (MammaPrint) and DNA (BRCA1/2, CVD multi-gene assay) levels supported the decision to perform extended genetic testing using WES. BMI: body mass index; CYP: cytochrome P450.

Figure 2

Detection of the RAD50 missense mutation R385C (rs139372231, Refseq mRNA accession NM_005732.3) using whole exome sequencing (A); and visualized using the Integrative Genome Viewer software tool [36]. The C to T base change at nucleotide position 1153 was confirmed by Sanger sequencing (B) as indicated by the arrow.

Figure 3

Detection of the MUC1 missense mutation Q85E (rs773704188, Refseq mRNA accession NM_002456.5) using WES (A); and visualized using the Integrative Genome Viewer software tool [36]. The C to G base change at nucleotide position 325 was confirmed by Sanger sequencing (B) as indicated by the arrow.

Figure 4

Pedigree of the index patient (arrow) subjected to whole exome sequencing, depicting the clinical (age of onset), pathology (tumour characteristics) and genetic heterogeneity in the family. Homozygosity for a minor allele is indicated by (+/+) and heterozygosity by (+/−). ER+: estrogen receptor-positive.

Figure 5

A simplified representation of the data analysis pipeline used by Torrent Suite for the identification of variants in WES data. Base calling, read filtering and trimming were performed by the Torrent Suite software. The torrent mapping alignment (TMAP) [60] program was used to map the resulting reads to the human reference genome. Variant calling was performed by the Torrent Variant Caller (TVC) in three steps: generate candidates, evaluate candidates and filter candidates. Final filtering and annotation of variant call files (VCF) were performed using GeneTalk [62] web-based bioinformatics tool.