Genetic inactivation of FAAP100 causes Fanconi anemia due to disruption of the monoubiquitin ligase core complex

The Fanconi anemia/breast cancer (FA/BRCA) DNA repair network promotes the removal of DNA interstrand crosslinks (ICLs) to counteract their devastating consequences, including oncogenesis. Network signaling is initiated by the FA core complex, which consists of 7 authentic FA proteins and an FA-associated protein, FAAP100, with incompletely characterized roles and unknown disease associations. Upon activation, the FA core complex functions as a multiprotein E3 ubiquitin ligase centered on its catalytic module, the FANCB-FANCL-FAAP100 (BLP100) subcomplex, for FANCD2 and FANCI monoubiquitylation. Here, we identified a homozygous variant in FAAP100, c.1642A>C, predicting p.(T542P), in a fetus with malformations suggestive of FA. The mutation caused sensitivity to ICL-inducing agents in cells from the affected individual and genetically engineered, FAAP100-inactivated human, avian, zebrafish, and mouse cells. All FAAP100-deficient cell types were rescued by ectopic expression of WT FAAP100, but not FAAP100T542P. In a confirmatory animal model, customized Faap100-/- mice exhibited embryonic lethality, microsomia, malformations, and gonadal atrophy resembling mice with established FA subtypes. Mechanistically, FAAP100T542P impaired ligase activity by preventing BLP100 subcomplex formation, resulting in defective FAAP100T542P nuclear translocation and chromatin recruitment. FAAP100 dysfunction that disrupted the FA pathway and impaired genomic maintenance, together with FA-consistent human manifestations, recommends FAAP100 as a legitimate FA gene, alias FANCX.