Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia
Kerstin Knies, Shojiro Inano, María J. Ramírez, Masamichi Ishiai, Jordi Surrallés, Minoru Takata, Detlev Schindler
Kerstin Knies, Shojiro Inano, María J. Ramírez, Masamichi Ishiai, Jordi Surrallés, Minoru Takata, Detlev Schindler
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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 5

Characterization of an Rfwd3–/– mouse model.

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Characterization of an Rfwd3–/– mouse model. (A) Numbers and proportions...
(A) Numbers and proportions of female (red) and male (blue) Rfwd3+/–, Rfwd3–/–, and Rfwd3+/+ mouse offspring of heterozygous mating. Rfwd3–/– pups were born at a sub-Mendelian rate. (B) Left panel displays atrophic Rfwd3–/– mouse testis. Remaining seminiferous tubules have little active spermatogenesis and few spermatozoa. Right panel shows Rfwd3+/+ testis with normal spermatogenesis for control. The original micrographs were taken at ×40. (C) Left panel displays atrophic Rfwd3–/– mouse ovary. There are clusters of luteal cells within a delicate stroma, but there is no follicular development. Right panel shows Rfwd3+/+ ovary with normal follicular development for control. The original micrographs were taken at ×40. (D) Cell cycle analysis examining G2-phase arrest in Rfwd3–/– versus Rfwd3+/+ MEFs without or with exposure to MMC. Increased G2 compartment size is highlighted in red, normal size shown in gray. (E) Histograms reflecting proportions of cells with the indicated number of chromosomal breaks per metaphase, blue without, red after exposure to MMC, in Rfwd3–/– and Rfwd3+/+ MEFs. Fifty cells each were scored. Sensitivity is indicated by the high rate of metaphases with more than 7 breaks (dashed line). Fifty metaphases each were scored. (F) Micrographs of Rfwd3–/– MEF metaphase preparations. Unaffected cell without prior exposure to MMC (top left), increased chromosomal breakage after exposure to MMC (bottom left), and predominance of chromatid-type lesions and radial reunion figures (arrows, right) after exposure to MMC. The original micrographs were taken at ×100. (G) Dose-response curves of Rfwd3–/– MEFs, Rfwd3+/+ MEFs, and Rfwd3–/– MEFs transduced with human WT-RFWD3 exposed to MMC. The data represent mean ± SEM; N = 3.