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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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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.
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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.

Mentions: To prove that the mutations identified in SLX4 were causal for the FA phenotype of both patients, we introduced the WT or the mutant SLX4 cDNAs into the patients’ fibroblasts (RA3083 and RA3331) and performed functional complementation assays (Figure 3 and Figure S4). Expression of WT SLX4 in both cell lines almost fully rescued the MMC sensitivity (Figure 3A, Figure S4A, and S4B), the late S/G2 arrest with MMC treatment (Figure 3B, Figure S4C, S4D, and S4E), and the chromosomal instability after treatment with DEB (Figure S4F). Some residual MMC sensitivity, cell cycle arrest, and chromosomal breakage is most likely due to some cells losing expression of the SLX4 as evident by immunofluorescence analysis (data not shown). Introduction of the mutant proteins did not rescue the FA phenotypes of the patients’ cells although a slight improvement was noted in the various assays possibly due to overexpression of the mutant proteins, which might have residual function. These experiments demonstrate that biallelic SLX4 mutations cause a new subtype of Fanconi anemia, FA-P and FANCP becomes an alias for the SLX4 gene.


Mutations of the SLX4 gene in Fanconi anemia.

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3345287&req=5

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.
Mentions: To prove that the mutations identified in SLX4 were causal for the FA phenotype of both patients, we introduced the WT or the mutant SLX4 cDNAs into the patients’ fibroblasts (RA3083 and RA3331) and performed functional complementation assays (Figure 3 and Figure S4). Expression of WT SLX4 in both cell lines almost fully rescued the MMC sensitivity (Figure 3A, Figure S4A, and S4B), the late S/G2 arrest with MMC treatment (Figure 3B, Figure S4C, S4D, and S4E), and the chromosomal instability after treatment with DEB (Figure S4F). Some residual MMC sensitivity, cell cycle arrest, and chromosomal breakage is most likely due to some cells losing expression of the SLX4 as evident by immunofluorescence analysis (data not shown). Introduction of the mutant proteins did not rescue the FA phenotypes of the patients’ cells although a slight improvement was noted in the various assays possibly due to overexpression of the mutant proteins, which might have residual function. These experiments demonstrate that biallelic SLX4 mutations cause a new subtype of Fanconi anemia, FA-P and FANCP becomes an alias for the SLX4 gene.

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