Multiple gene sequencing for risk assessment in patients with early-onset or familial breast cancer

Abstract

Since BRCA mutations are only responsible for 10-20% of cases of breast cancer in patients with early-onset or a family history and since next-generation sequencing technology allows the simultaneous sequencing of a large number of target genes, testing for multiple cancer-predisposing genes is now being considered, but its significance in clinical practice remains unclear. We then developed a sequencing panel containing 68 genes that had cancer risk association for patients with early-onset or familial breast cancer. A total of 133 patients were enrolled and 30 (22.6%) were found to carry germline deleterious mutations, 9 in BRCA1, 11 in BRCA2, 2 in RAD50, 2 in TP53 and one each in ATM, BRIP1, FANCI, MSH2, MUTYH, and RAD51C. Triple-negative breast cancer (TNBC) was associated with the highest mutation rate (45.5%, p = 0.025). Seven of the 9 BRCA1 mutations and the single FANCI mutation were in the TNBC group; 9 of the 11 BRCA2, 1 of the 2 RAD50 as well as BRIP1, MSH2, MUTYH, and RAD51C mutations were in the hormone receptor (HR)(+)Her2(-) group, and the other RAD50, ATM, and TP53 mutations were in the HR(+)Her2(+) group. Mutation carriers were considered as high-risk to develop malignancy and advised to receive cancer screening. Screening protocols of non-BRCA genes were based on their biologic functions; for example, patients carrying RAD51C mutation received a screening protocol similar to that for BRCA, since BRCA and RAD51C are both involved in homologous recombination. In conclusion, we consider that multiple gene sequencing in cancer risk assessment is clinically valuable.

Keywords: BRCA; genetic counseling; hereditary breast cancer; multiple gene sequencing; variant of uncertain significance.

Figure 1. Mutation of predisposing genes in breast cancer patients with early-onset or a family history

(A) Each of the predisposing genes identified in the patients is listed on the left. A family history is shown in black. For the molecular types, HR(+)Her2(−) breast cancer is colored pink, HR(+)Her2(+) orange, HR(−)Her2(+) light green, and TNBC purple. (B) Twenty-five mutations of a predisposing gene were identified, 1 (4.0%) in ATM, 8 (32.0%) in BRCA1, 10 (40.0%) in BRCA2, and 1 each (4.0%) in BRIP1, FANCI, MSH2, RAD50, RAD51C, and TP53.

Figure 2. Structural analyses of three mutations

(A) Ribbon presentation of the FANCI E96K mutant structure (dark blue) superimposed on the structure of the wild type FANCI interacting with FANCD2 (FANCI, light blue; FANCD2, purple; pdb 3S4W) to form the ssDNA groove. This interface is maintained by Van Der Walls forces between FANCD2 HD2 (Leu614) and FANCI solenoid 1 (Leu92, Met94, and Leu95). The lysine substitution, with a longer sidechain, may disrupt the FANCI cap-solenoid 1 structure, leading to disturbance of the binding affinity for FANCD2 and influencing the ssDNA groove. This may result in defective function of the FANCI-FANCD2 complex and inability to carry out DNA interstrand cross-linking. FANCD2 Lys559 (yellow) is a mono-ubiquitination site. (B) Ribbon presentation of the MSH2 R534L structure (red) superimposed on that for the wild type MSH2-MSH6 complex (pdb: 2O8E, light gray; MSH6, light blue; DNA helix, dark blue), showing that MSH2 Arg534 is located at the clamping region, which is involved in DNA contact and matching of MSH6. Substitution of the arginine with leucine reduces the basic nature of this region, which may alter the attraction of DNA. This structure change also affects the interaction with MSH6. MSH2 p. R534L is highly suspected to have decreased function. (C) PMS2 p. R295W (khaki color) superimposed on the wild type PMS2 structure (pdb: 1EA6, light blue). This missense mutation changes the polar amino acid arginine to the non-polar tryptophan and is located nearby the entrance to the ATP binding pocket. However, whether this causes a functional defect requires further functional assays.