Mutations of the SLX4 gene in Fanconi anemia

Abstract

Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.

Figure 1

Characterization of cell lines from FA patients with SLX4 mutations. A. Western blot analysis with an anti-FANCD2 antibody of U2OS cells transfected with the indicated siRNAs and treated with 1 µM MMC for 24 hours. L (long) indicates a monoubiquitinated form and S (short) indicates the non-monoubiquitinated form of FANCD2. B. RT-qPCR in U2OS cells transfected with various siRNAs against SLX4 used in experiment shown in A. Error bars indicate standard deviation of three replicates. C. Western blot analysis with an anti-FANCD2 antibody of BJ, RA3083 and RA3331 fibroblasts. Cells were left untreated or were treated with 1 µM MMC for 24 hours. D. Pedigrees of the two families described in this study showing accession numbers for cell lines (RA) and peripheral blood samples (B, RB). The two probands are indicated with filled symbols. Mutation carriers are indicated by half-filled symbols. E. Examples of metaphases of the LCL RA3042 (no drug treatment) and fibroblast RA3083 cell lines from the patient 1084/1 and the fibroblasts RA3331 from patient 414/1 (treatment with diepoxybutane (DEB)).

Figure 2

SLX4 is defective in two FA patients. A. Schematic of SLX4 (based on ref. 7) showing the domain architecture, the interacting proteins, and the predicted protein effect of SLX4 mutations in IFAR1084/1 and IFAR414/1 patients. B. Analysis of the mutant SLX4 protein in the patient’s cell lines. Cell extracts of primary BJ, RA3083, and RA3331 fibroblasts were subjected to immunoprecipitation using a control rabbit antibody (control IgG) or the SLX4 antibody. Asterisks indicate the crossreacting bands. Note that the antibody does not identify SLX4 in straight western (lanes 7 to 9).

Figure 3

Complementation of RA3083 and RA3331 cells with the SLX4 cDNA. A. Complementation of MMC sensitivity. Fibroblasts stably transduced with empty vector (control), or the vector expressing WT SLX4 or the mutant SLX4 cDNAs were exposed in triplicate to different levels of MMC ranging from 0 to 100 nM. After 8 days, cell number was determined using a coulter counter. Total cell numbers at each dose were divided by the number of cells in the untreated sample to arrive at percent survival. Error bars indicate standard deviation. B. Complementation of the cell cycle defect after MMC treatment. Indicated cells were treated with 100nM MMC and the cell cycle was analyzed 48 hours later. Untreated samples were analyzed in parallel. Expression levels of the exogenous proteins are shown in Figure S4A, S4B, and S4C. Quantification of the data is shown in Figure S4D and S4E.

Figure 4

Interaction of mutant forms of SLX4 with its partners and with ubiquitin. A. Analysis of SLX4 interacting partners in SLX4 mutant cells. Cell extracts of primary fibroblasts (BJ, RA3083, and RA3331) were subjected to immunoprecipitation using the SLX4 antibody. Interacting proteins were identified by immunoblotting with the indicated antibodies. B. Analysis of SLX4 interacting partners in RA3331 cells. Cell extracts of RA3331 E6E7 cells expressing HA tagged control vector (CONTROL), WT SLX4, or the p.L672VfsX119 SLX4 were subjected to immunoprecipitation using HA antibody or HA antibody in the presence of 30 µg of HA peptide. Interacting proteins were identified by immunoblotting with the indicated antibodies. C. Interaction of the UBZ domains with ubiquitin. GST-purified GST control, WT UBZ domains (SLX4aa 251-402) and UBZ domains with four cysteines mutated to alanines (SLX4aa251-402_C296A_C299A_C336A_C339A) were incubated with the indicated forms of ubiquitin, purified by binding to GST beads, separated on a PAGE gel and immunobloted with anti-ubiquitin antibody. The bottom panel shows Ponceau staining of the GST proteins.