The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM ^-/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors.

Keywords: Cancer genetics.

Fig. 1

Functional studies of the FANCM:p.Arg658*, p.Gln1701* and p.Arg1931* truncating variants using the patient-derived FANCM^−/− EGF280 cell line. a Western blot showing the FANCM expression in EGF280 cells complemented with lentiviral vectors harboring the three different variants. Bands corresponding to truncated FANCM protein were visible for EGF280 + p.Gln1701* and p.Arg1931*, and no bands were present for the EGF280 + p.Arg658*. b Study of the expression of the FANCM protein in EGF280 + p.Arg658*. The c.1972C > T base substitution, causing the p.Arg658* variant abrogates a digestion site for the restriction enzyme TseI that is present in the wild-type (wt) cDNA sequence. Total RNA was extracted from EGF280 + wtFANCM and from the EGF280 + p.Arg658* and subjected to reverse transcription. PCR-amplified cDNA products were digested with TseI. Digested and undigested cDNAs were loaded. In the first two lanes are shown bands of 386 bp corresponding to uncut wt cDNA, and bands of 257 and 129 bp corresponding to cut wt cDNA. In next two lanes bands of 386 bp indicate that p.Arg658* cDNA was not cut due to the c.1972C > T base substitution abrogating the TseI site. In the two lanes after the molecular weight marker (M) undigested and digested products of the two previous PCR products were mixed 1:1 and loaded as a control. c Analysis of diepoxybutane (DEB) sensitivity on cell survival. The EGF280 cells expressing p.Arg658* are significantly more sensitive to DEB than the cells expressing p.Gln1701* or p.Arg1931* (P-values from Tukey’s range test are reported in Supplementary Table 4). EGF280 and EGF280 + wtFANCM are used as controls (N = 3; error bars: standard deviation). d Chromosome fragility induced by DEB treatment (100 ng/ml). Here, the chromatidic break patterns of the cells expressing wt FANCM, of the cells harboring p.Gln1701* or p.Arg1931* variants, and of the native EGF280 cells or the cells expressing p.Arg658* were statistically different. (P-values from chi-squared test; N = 2). e Analysis of cellular sensitivity to olaparib. Contrarily to what we observed in the DEB sensitivity assays, survival rates of the different complemented cell lines were apparently not different. Human fibroblasts (BRCA2^−/−) were homozygous for the c.469 A > T (p.Lys157*) truncating variant and were used as a positive control. (P-values from Tukey’s range test are reported in Supplementary Table 5; N = 3; error bars: standard deviation). All blots derive from the same experiment and were processed in parallel

Fig. 2

Schematic diagram of the 2,048 amino acid long FANCM protein. The functional or binding domains (BD) are indicated in black and as reported in Deans and West, 2009. The position of the three FANCM truncating variants c.1972C > T (p.Arg658*), c.5101 C > T (p.Gln1701*) and c.5791 C > T (p.Arg1931*) is also shown