The genomic region of SLX4
, comprising all exons and exon-intron boundaries, was sequenced in 94 BrCa familial cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1
mutations (see Methods). This mutational analysis revealed 49 variants: 21 novel and 28 which are currently annotated in the single nucleotide polymorphism database (dbSNP [23
]) (Table ). Of the 49 variants, 21 were found only once, which include three changes identified by the 1,000 Genomes Project [24
] (Table ): rs72778139-T has no known frequency data; rs76488917-A has an allele frequency of 0.02 in Caucasians; and rs115694169-A has an allele frequency of 0.03 in the Yoruba people of Ibadan (there is no data for Caucasians). Excluding these from the set of 21 with low frequency revealed eight missense and five silent changes at the protein level, and five intronic changes (Table ). A neural network splicing prediction [25
] model did not strongly support alteration by any of the identified intronic variants (data not shown). Together, these results suggest extensive genetic variation at the SLX4
locus among individuals in our population, but provide no obvious link to BrCa risk.
SLX4 variants found in non-BRCA1/2-mutated familial BrCa cases
Having identified rare variants in BrCa familial cases, we next assessed the presence of 10 of these variants in a cohort of controls collected at the same hospital as the cases (see Methods). The selection of these variants was based on the observed low frequency in the 94 BrCa cases and on their identification as missense variations. In addition, a causative prediction was obtained using two algorithms (PolyPhen-2 [26
] and SIFT [27
]), plus a weighted average of scores (Condel [28
]). Seven of these variants were not found in controls and, intriguingly, five of them were predicted to be "deleterious" (Table ). Among this group, only one amino acid position (Trp823) showed some evolutionary conservation (Figure ), and the substitution may be disfavored (Trp to Cys) [29
]; tumor samples were not available for any case that would have allowed assessment of the existence of loss of heterozygosity at the SLX4
locus. Nonetheless, predictions of a deleterious effect should be taken with caution as neutral polymorphisms can frequently be misclassified (from ~15-50% depending on the method [28
]). On the other hand, extensive genetic variation in SLX4
might reflect an unknown evolutionary pressure or could be related to a similar observation made for other DNA repair-related genes [30
Pathological prediction and frequency in controls of selected SLX4 missense variants
CLUSTALW-based multi-alignment of human SLX4 and eukaryotic homologs. The region surrounding human Trp823 is shown.
While SLX4 serves as a scaffold for multiple proteins involved in the DNA damage response [16
], the functional involvement of the Trp823 position, and of the other rare variants not found in controls in this study, is unknown. None of the identified variants changes a critical amino acid residue and there is no data that could suggest an alteration of protein interactions or complexes; however, the Pro1470Leu variant might disrupt a mitotic phosphorylation site at Ser1469 [31
]. In this context, a SLX4
pathological variant linked to BrCa should consist of a hypomorphic mutation that would cause genome instability. Accordingly, SLX4 is a key regulator of the function of structure-specific endonucleases involved in the repair of DNA damage; in particular, proper function of SLX4 is fundamental for repair during replication and for resolving Holliday junctions formed during homologous recombination [16
]. Overall, the results of this study do not support the existence of loss-of-function mutations of SLX4 associated with BrCa risk; nonetheless, further genetic analysis in patients and controls, combined with functional assays of specific rare variants, may be warranted.