FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Abstract

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Figure 1.

Sequencing analysis of the FANCM gene and transcript. (A) Genomic DNA fragment PCR amplified using both primers in exon 22 from an LCL carrying the c.5791C>T mutation. (B) cDNA fragment amplified by PCR using a forward primer in exon 22 and a reverse primer in exon 23. A strong reduction in the expression of the mutant allele was observed in both cycloheximide treated and untreated cells. cDNA from a non-carrier individual was used as a control. The position of the mutation is indicated by the arrows. Identical results were observed in an additional mutated LCL.

Figure 2.

Analysis of the effect of the FANCM c.5791C>T mutation on RNA. (A) Sequence logo of hnRNP A1 binding sites generated as described in Materials and Methods. The opal codon (TGA, boxed) introduced by the FANCM c.5791C>T mutation (in bold red) is contained at positions 1–3 of the hnRNP A1 binding-site encompassing nucleotides c.5790_5795. The hnRNP A1 binding-site strength computed by the ASSEDA software for the normal and mutated sequences is reported. (B) Agarose gel electrophoresis of the RT-PCR products using a forward primer in exon 21 and a reverse primer in exon 23. M, molecular marker (ΦX-174 HaeIII digested); 1, no template as a negative control for PCR; 2, genomic DNA as a negative control for the specificity of cDNA amplification; 3 and 4, cDNAs from LCLs carrying the c.5791C>T mutation; 5–14, cDNAs from LCLs derived from 10 mutation negative individuals, used as reference controls. The sizes of the full-length (FL) and Δexon22 (Δ22) transcripts are indicated. (C) Sequence of the aberrant band excised from the gel showing the skipping of the entire exon 22. (D) Western blot analysis of biotin RNA–hnRNP A1 protein pull down using a goat polyclonal antibody. The sequence of the used RNA oligonucleotides encompassing FANCM positions r.5779_5804 is reported, with the r.5791C>U mutation in bold red, the opal codon enlightened in light grey and the predicted hnRNP A1 binding site created by the mutation boxed in red. As a control for the pull-down efficiency and specificity, we used an antibody against the ELAVL1/HuR protein for which a binding site, boxed in green, is predicted in both RNA oligonucleotides. Input, 10% of total HeLa cell line extract used in the pull-down assay. No, no RNA used as a negative control; 1, normal RNA; 2, RNA carrying the r.5791C>U mutation. The results shown here are representative of two independent experiments.

Figure 3.

Functional studies of the FANCM mutation. (A) Analysis of cellular MMC sensitivity. MEFs expressing Δ22-FANCM allele (Fancm^−/− + Δ22 FANCM) are more sensitive to MMC than the cell expressing the wt FANCM allele (Fancm^−/− + wt FANCM). Not transduced MEFs (MEF Fancm^−/−) and MEF Fancm^−/− expressing a FANCM with loss-of-function mutation p.S724X (Fancm ^−/− + stop FANCM) are used as controls (N = 3; error = standard deviation). (B) Chromosome fragility induced by DEB treatment. Fancm^−/− + Δ22 FANCM and Fancm^−/− + stop FANCM cells show higher chromosome fragility than Fancm^−/− + wt FANCM. Twenty metaphases were analysed for chromosome breaks. Results are represented as mean number of breaks per cells and the error bars are SEM.