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 3

Functional analyses of ΔRFWD3 DT40 cells.

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Functional analyses of ΔRFWD3 DT40 cells. (A) Frequency of gene targetin...
(A) Frequency of gene targeting at the ovalbumin (OVA) and KU70 loci of WT DT40 cells (blue) and 2 independently generated ΔRFWD3 DT40 cell lines (#1, red; #2, green). Percentages of the targeting events relative to the number of examined clones are shown on top of each bar. (B) Frequency of neo-resistant DT40 colonies due to HR events in the SCneo recombination substrate integrated in the OVA locus. Cells with indicated genotypes were transiently transfected with empty vector (+Vector) or with vector containing I-SceI (+I-SceI) and selected in medium containing G418. After 10–14 days the number of colonies was counted. (C and D) Dose-response curves of WT DT40 cells versus ΔRFWD3 DT40 cells and ΔRFWD3 DT40 cells transfected with WT-RFWD3, RFWD3-I615K, or RFWD3-C267A. Cells were exposed to MMC or cisplatin. (E) Histograms reflecting proportions of cells with the indicated number of chromosomal lesions per metaphase in WT DT40 and mutant cells with the indicated genotypes and transfections without or with exposure to MMC. Fifty cells each were scored. (F) GFP immunofluorescence analysis of ΔRFWD3 DT40 fibroblasts transiently transfected with mock, GFP-chRFWD3-C267A, or the double mutant GFP-chRFWD3-C267A/I615K after exposure to MMC. (G) Proportion of GFP-RFWD3–positive ΔRFWD3 DT40 cells (top panel) and GFP-RFWD3 foci–positive ΔRFWD3 DT40 cells (>5, middle panel) and number of GFP-RFWD3 foci in individual GFP-RFWD3 foci–positive ΔRFWD3 DT40 cells (bottom panel) after transfection with the indicated plasmids. Cells are those used in F. (H) Relocation of GFP-RFWD3 in ΔRFWD3 DT40 cells transfected with the indicated plasmids without and with exposure to MMC. Fractions were probed using anti-GFP and anti–histone 3 antibodies. Data in B–D and G represent mean ± SEM; N = 3. Significance in G determined by unpaired, 2-tailed Student’s t test. NS, not significant.