Establishment Of Molecular Diagnosis Platform Of Fanconi Anemia And The Role Of NEIL3 In DNA Inter-strand Crosslink Repair

Section I.Establishment of a molecular diagnostic platform for Fanconi anemiaFanconi anemia(FA)is a rare recessive disease characterized by progressive bone marrow failure,congenital abnormalities,and increased incidence of cancers.To date,mutations in 22 genes can cause FA or an FA-like phenotype.In China,in addition to clinical information,FA diagnosis primarily relies on genetic sequencing since the chromosome breakage test is rarely performed.Here,we employed multiple genetic diagnostic tools(DNA sequencing,multiplex ligation-dependent probe amplification,and chromosome microarray)as well as a variant-based functional assay platform to investigate the genetic cause in 28 suspected Chinese FA patients.A total of 49 distinct candidate variants were detected in 6 FA genes(36 variants in FA-A,1 variants in FA-B,3 variants in FA-C,2 variants in FA-D2,5variants in FA-G,and 2 variants in FA-J),of which,40 were novel.Eight missense and one indel variants were unable to restore FANCD2 mono-ubiquitination and MMC resistance in a panel of FA indicator cell lines,indicating that these mutations are deleterious.Three missense variants(FANCA-L424 V,FANCC-E273 K,and FANCG-A153G)were harmless.Finally,27 patients were molecularly diagnosed with FA,consistent with their clinical phenotype.In the FA-A subgroup,large deletions accounted for 20% of the disease-causing variants.We have established a comprehensive molecular diagnostic workflow for Chinese FA patients,which can substitute for standard FA cytogenetic analysis.Section ?.The role of NEIL3 in Interstrand Crosslink RepairThe NEIL3 DNA glycosylase excises bulky lesions from DNA including interstrand crosslinks(ICL).Although NEIL3 has been shown to unhook psoralen-induced ICL in Xenopus extracts,how NEIL3 participants in ICL repair in human cells and its corporation with the classic Fanconi anemia(FA)/BRCA pathway remains unclear.Here we show that NEIL3 is the major pathway for repairing psoralen-ICL,and FA/BRCA pathway is only activated when NEIL3 is not available.Mechanistically,NEIL3 is recruited to psoralen-ICL in a rapid,PARP-dependent manner.The NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks(DSBs),unlike the FA/BRCA pathway.The RUVBL1/2 complex physically interacts with NEIL3 and functions within the NEIL3 pathway in psoralen-ICL repair.TRAIP is important for the recruitment of NEIL3 but not FANCD2,and knockdown of TRAIP promotes FA/BRCA pathway activation.However,TRAIP is synthetically lethal with both NEIL3 and FA pathways upon PUVA treatment,suggesting that TRAIP may function upstream of the two pathways and may influence pathway choice.Taken together,NEIL3 is the major pathway for repairing psoralen-ICL through its unique DSB-free mechanism in human cells.