Typer software version 4.0

Genomic DNA was isolated from peripheral blood lymphocytes by using a Quick Gene DNA Whole Blood Kit S (Thermo Scientific, USA) according to the manufacturer’s protocol, and stored at −80 °C. The SNPs were genotyped by using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrophotometry to detect allele-specific primer extension products with the MassARRAY platform (Sequenom, Inc., USA). Assay data were analyzed using Sequenom TYPER software (version 4.0).

Associations between the selected variants and breast cancer were evaluated in further studies. Three variants were genotyped in a set of 250 SBC patients and 248FNCCs. Genotyping of the variants c.339G>A and c.973G>A was performed with the MassARRAY platform (Sequenom, San Diego, CA, USA) using the iPLEX Gold Assay. The amplification and its extended primers were designed by MassARRAY Designer of Sequenom. The information on the primers was listed in Table 2. The amplification reaction conditions were as follows: an initial denaturation at 94°C for 15 min, followed by 45 cycles of denaturing at 94°C for 20 s, annealing at 56°C for 30 s, and the extension at 72°C for 60 s; finally, the reaction was elongated at 72°C for 3 min. Reaction parameters of single-base extension were an initial incubation at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s with 5 nested cycles of 52°C for 5 s and 80°C for 5 s, respectively. Finally, singe-base extension was completed at 72°C for 3 min. Experimental data were analyzed by Typer software version 4.0 (Sequenom, San Diego, CA, USA). Genotyping of the variants c.51G>C and c.758C>A was done by PCR-sequencing assay. The primers and reaction conditions were the same as those used in the mutation screening for FANCC gene exon1 and exon7.