Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia
Kerstin Knies, … , Minoru Takata, Detlev Schindler
Kerstin Knies, … , Minoru Takata, Detlev Schindler
Published July 10, 2017
Citation Information: J Clin Invest. 2017;127(8):3013-3027. https://doi.org/10.1172/JCI92069.
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Research Article Genetics

Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia

Abstract

The WD40-containing E3 ubiquitin ligase RFWD3 has been recently linked to the repair of DNA damage by homologous recombination (HR). Here we have shown that an RFWD3 mutation within the WD40 domain is connected to the genetic disease Fanconi anemia (FA). An individual presented with congenital abnormalities characteristic of FA. Cells from the patient carrying the compound heterozygous mutations c.205_206dupCC and c.1916T>A in RFWD3 showed increased sensitivity to DNA interstrand cross-linking agents in terms of increased chromosomal breakage, reduced survival, and cell cycle arrest in G2 phase. The cellular phenotype was mirrored in genetically engineered human and avian cells by inactivation of RFWD3 or introduction of the patient-derived missense mutation, and the phenotype was rescued by expression of wild-type RFWD3 protein. HR was disrupted in RFWD3-mutant cells as a result of impaired relocation of mutant RFWD3 to chromatin and defective physical interaction with replication protein A. Rfwd3 knockout mice appear to have increased embryonic lethality, are subfertile, show ovarian and testicular atrophy, and have a reduced lifespan resembling that of other FA mouse models. Although RFWD3 mutations have thus far been detected in a single child with FA, we propose RFWD3 as an FA gene, FANCW, supported by cellular paradigm systems and an animal model.

Authors

Kerstin Knies, Shojiro Inano, María J. Ramírez, Masamichi Ishiai, Jordi Surrallés, Minoru Takata, Detlev Schindler

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Figure 2

Involvement of RFWD3 in HR and ICL repair.

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Involvement of RFWD3 in HR and ICL repair. (A) Reduced HR in RFWD3-defic...
(A) Reduced HR in RFWD3-deficient human cells as signaled by the I-SceI–induced HR assay. Shown is the decrease of GFP-positive (HR-active) cells compared with controls. The left graph shows data from siRNA-transfected U2OS cells (luciferase [Luc; mock] vs. BRCA2 and RFWD3). The middle graph represents data from non-FA versus RFWD3-mutant 1143 and BRCA2/FANCD1-mutant fibroblasts, a disease-control FA-D1 line with the homozygous mutation c.469A>T (p.Lys157*). The right graph displays data from 1143 transduced with WT-RFWD3 versus mock, I639K, and nontransduced 1143 fibroblasts. All results are corrected for transfection rate and size of S phase. (B) Western blot with RFWD3 antibody including lysates from 1143, her family members, and non-FA fibroblasts exposed to MMC. Lanes were run on the same gel but were noncontiguous. (C) Cell fractionation of protein lysates from non-FA and 1143 fibroblasts exposed to MMC. (D) Proportion of RPA1, RPA2, and RAD51 foci–positive cells in 1143, 1143+WT-RFWD3–transduced, and non-FA fibroblasts at different intervals after an initial 8-hour pulse of MMC exposure. (E) Dose-response curves of CRISPR clone CR21F5 versus parental U2OS cells, HAP1-RFWD3– versus HAP1 control cells, and 1143 versus non-FA fibroblasts exposed to MMC. The 1143 and non-FA curves are reused in Figure 1D. (F and G) Cell cycle analysis regarding G2-phase arrest of CRISPR clone CR21F5 versus parental U2OS cells and of HAP1-RFWD3– versus HAP1-RFWD3–+WT–complemented and HAP1 control cells without or with exposure to MMC. Increased G2 compartment size is highlighted in red; normal size is shown in gray. Data in A, D, and E represent mean ± SEM; N = 3 for siRNA experiments, otherwise N = 5. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired, 2-tailed Student’s t test.