Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families

BMC Cancer Analysis of SLX4/FANCP in non-BRCA1/2-mutated breast cancer families Juana Fernndez-Rodrguez 0 Francisco Quiles 0 Ignacio Blanco 0 Alex Teul 0 Ldia Feliubadal 0 Jess del Valle 0 Mnica Salinas 0 ngel Izquierdo Esther Darder Detlev Schindler Gabriel Capell 0 Joan Brunet Conxi Lzaro 0 Miguel Angel Pujana 1 0 Hereditary Cancer Program, Catalan Institute of Oncology (ICO), Hospital Duran i Reynals, Bellvitge Institute for Biomedical Research (IDIBELL) , L'Hospitalet, Barcelona, Catalonia , Spain 1 Translational Research Laboratory, Biomedical Research Center Network for Epidemiology and Public Health (CIBERESP) , ICO, IDIBELL, L'Hospitalet, Barcelona, Catalonia , Spain Background: Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results. Methods: The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations. Results: This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. Conclusions: Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease. - Background A functionally coherent network of gene and/or protein interactions, altered in Fanconi anemia (FA) and breast cancer (BrCa), has emerged in recent years [1]. Fifteen genes that, when mutated, cause FA (FANC genes) and several genes that may harbor mutations of high, moderate or low penetrance for BrCa risk encode for proteins that converge on a homology-directed DNA damage repair process [2]. As further evidence of a fundamental common causal basis between these diseases, germline bi- and mono-allelic loss-of-function mutations in four of these genes cause FA and BrCa, respectively: FANCD1/BRCA2 [3,4], FANCJ/BRIP1 [5-8], FANCN/ PALB2 [9-11] and FANCO/RAD51C [12,13] (more recent data suggests that mutations in RAD51C may be primarily linked to ovarian cancer risk [14]). This evidence marks any novel gene involved in the aforementioned network or process as a candidate to harbor mutations in unclassified FA and/or BrCa patients. Interstrand DNA cross-link agents, such as mitomycin-C used in diagnostic tests for FA, block replication forks and may therefore cause genome instability. Homologs of SLX4 in model organisms were initially identified as necessary for replication fork restart following exposure to DNA-damaging agents [15]. Subsequently, SLX4 homologs have been shown to play a key role as docking molecules for the repair of interstrand cross-links [16,17]. These observations pointed to the human SLX4 gene as a FANC candidate for unclassified patients and, as a result, two groups have recently described mutations (renamed FANCP gene or FA-P subtype) [18,19]. The potential link to BrCa risk has been examined, to date, in 52 German or Byelorussian patients with familial breast cancer [20]: the study has not revealed truncating or clearly pathogenic mutations, but has identified four unclassified missense variants. Here, we conducted a more detailed study of the SLX4 gene in 94 index BrCa cases from Spanish families negative for BRCA1 and BRCA2 mutations. As recently reported [20], our results do not show truncating or clearly pathogenic mutations, although they do describe seven missense variants of unknown biological significance that are not found in controls. Methods Study samples Since its creation in 1999, the Hereditary Cancer Program at the ICO has identified a set of high-risk families with suspected hereditary breast and/or ovarian cancer syndrome. Following the Catalan Consensus Onco Guidelines on genetic testing for this condition, patients are analysed for mutations in the BRCA1 or BRCA2 genes after receiving appropriate genetic counselling and providing written informed consent. This genetic analysis consists of screening for point mutations and large rearrangements affecting those genes. For the present study, a total of 94 affected individuals belonging to 94 unrelated families negative for BRCA1 or BRCA2 mutations were selected. In addition to negativity for mutations in BRCA1 or BRCA2, the inclusion criteria were: at least three first-degree relatives affected by breast or ovarian cancer; or at least two first-degree female relatives affected by breast cancer (at least one of them diagnosed before the age of 50); or at least one case of female breast cancer plus at least one case of either ovarian, female bilateral breast, or male breast cancer. Among the selected cases, 10 families were represented with an elevated prior risk of harboring a high-penetrance mutation, as calculated with the BRCAPRO [21] algorithm (briefly, 94 families mean = 0.40, standard deviation = 0.26, 95% confidence interval 0.11-0.99). Control samples, consisting of 138 women and 146 men, were taken from a hospital-based cancer association study (a detailed description of the study population, composition and interviews has been given elsewhere [22]). Specifically, these individuals were randomly enrolled from noncancer patients admitted to the same general hospital as the BrCa cases. To avoid selection bias, the inclusion criterion for controls was that the current admission to the hospital should be for a new disease (not previously diagnosed). The studies were approved by the IDIBELL ethics committee and participants gave written informed consent for their participation, and for the genetic analysis of their biological samples, according to the Declaration of Helsinki. Mutation analysis The SLX4 exons and exon-intron boundaries were sequenced from polymerase chain reactions using previously defined primers and conditions [18]; for exon 7, AmpliTaq Gold DNA Polymerase (Applied Biosystems) and 10% of dimethyl sulfoxide were used. The reaction products were purified from remaining primers using ExoSAP-IT (GE Healthcare) and sequencing reactions performed following a standard Sanger method with the BigD (...truncated)