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Mutations of the SLX4 gene in Fanconi anemia.

Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A - Nat. Genet. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

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.

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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)).
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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)).

Mentions: As depletion of SLX4 in a U2OS cell line does not affect FANCD2 ubiquitination (Figure 1A and B), we sequenced SLX4 in the families from the International Fanconi Anemia Registry16 with unassigned FA complementation groups and normal FANCD2 modification (Figure 1C) and identified two families carrying germline mutations, IFAR1084 and IFAR414 (Figure 1D). Phenotypes of the two patients are summarized in Table 1. The lymphoblastoid cell line (LCL) (RA3042) and fibroblasts (RA3083) from the patient 1084/1 showed increased genomic instability (Figure 1E and Table 2) and increased sensitivity to Mitomycin C (MMC) (Figure S1A). The 414/1 patient’s LCL (RA 1376) was not sensitive to MMC suggestive of reversion (Figure S1B); however, his skin fibroblasts (RA 3331) displayed a high degree of DEB induced chromosomal instability (Figure 1E and Table 2) and sensitivity to MMC. No UV sensitivity was observed in fibroblasts from either of the patients (Figure S1C and D). Fibroblasts from the patient 414/1 (RA3331), but interestingly not patient 1084/1 (RA3083), were sensitive to camptothecin (CPT), a topoisomerase I inhibitor (Figure S1E and F).


Mutations of the SLX4 gene in Fanconi anemia.

Kim Y, Lach FP, Desetty R, Hanenberg H, Auerbach AD, Smogorzewska A - Nat. Genet. (2011)

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)).
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Related In: Results  -  Collection

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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)).
Mentions: As depletion of SLX4 in a U2OS cell line does not affect FANCD2 ubiquitination (Figure 1A and B), we sequenced SLX4 in the families from the International Fanconi Anemia Registry16 with unassigned FA complementation groups and normal FANCD2 modification (Figure 1C) and identified two families carrying germline mutations, IFAR1084 and IFAR414 (Figure 1D). Phenotypes of the two patients are summarized in Table 1. The lymphoblastoid cell line (LCL) (RA3042) and fibroblasts (RA3083) from the patient 1084/1 showed increased genomic instability (Figure 1E and Table 2) and increased sensitivity to Mitomycin C (MMC) (Figure S1A). The 414/1 patient’s LCL (RA 1376) was not sensitive to MMC suggestive of reversion (Figure S1B); however, his skin fibroblasts (RA 3331) displayed a high degree of DEB induced chromosomal instability (Figure 1E and Table 2) and sensitivity to MMC. No UV sensitivity was observed in fibroblasts from either of the patients (Figure S1C and D). Fibroblasts from the patient 414/1 (RA3331), but interestingly not patient 1084/1 (RA3083), were sensitive to camptothecin (CPT), a topoisomerase I inhibitor (Figure S1E and F).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genome Maintenance, The Rockefeller University, New York, New York, USA.

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.

Show MeSH
Related in: MedlinePlus