Assessment of <i>SLX4</i> Mutations in Hereditary Breast Cancers

<div><p>Background</p><p><i>SLX4</i> encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in <i>SLX4</i> in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.</p><p>Methods and Results</p><p>To determine if <i>SLX4</i> is involved in breast cancer susceptibility, we sequenced the entire <i>SLX4</i> coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known <i>BRCA1</i> or <i>BRCA2</i> mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 <i>SLX4</i> variants (4 novel and 1 rare) cDNAs in a human <i>SLX4</i>-null fibroblast cell line, RA3331. While wild type <i>SLX4</i> and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* <i>SLX4</i> mutant failed to do so.</p><p>Conclusion</p><p>Loss-of-function mutations in <i>SLX4</i> may contribute to the development of breast cancer in very rare cases.</p></div

Identification of the nonsense<i>SLX4</i> variant and evaluation of pathogenicity of the <i>SLX4</i> variants by complementation assay.

<p>(A) Sanger sequence traces for DNA extracted from whole blood and breast tumor of the patient with the c.2469G>A (p.W823*) <i>SLX4</i> mutation (NCBI Refseq NM_032444.2). (B) Anti-HA immunoblot showing expression of HA-tagged SLX4 variants in RA3331/E6E7/hTERT cells. Alpha-tubulin serves as loading control. (C)∼(E) Cell survival assays of the RA3331/E6E7/hTERT cell lines expressing indicated SLX4 variants in response to MMC (C), CPT (D) and the PARP inhibitor, Olaparib (E). RA3331/E6E7/hTERT fibroblast cell lines expressing empty vector, wild type SLX4 and indicated SLX4 variants were treated in triplicate with increasing concentrations of MMC (0–100 nM), CPT (0–16 nM) and Olaparib (0–50 µM). After 8 days in culture, the cell number was determined using a Coulter counter. The number of cells at each drug concentration was divided by the number of cells in the untreated sample to calculate the percentage of cell survival. The error bars indicate standard deviations based on three replicates.</p