Molecular Characterization of Novel Mutations in Fanconi Anemia Patients

Fanconi anemia (FA) is a rare disorder that is characterized by bonemarrow failure in the first decade of life, developmental abnormalities, and predisposition to malignancies. The majority of patients have mutations in one of the 22 known FA genes, while a small number of patients have not been assigned to a complementation group. FA proteins are required for the proper repair of DNA interstrand crosslinks (ICL), a deleterious type of DNA damage that covalently binds DNA strands. We have used Whole Exome Sequencing (WES) in conjunction with cell-based assays to determine disease-causing mutations in a subset of patients enrolled in the International Fanconi Anemia Registry (IFAR) who are not assigned to a known complementation group. In this thesis, we present three cases that were the focus of study. We describe a new FA complementation group identified in a patient presenting with typical FA features and deficiency of the ubiquitin-conjugating enzyme (E2), UBE2T. No pathogenic gene variants were identified by WES, but RNA sequencing (RNA-seq) uncovered a significant decrease in UBE2T transcript, and western blot confirmed deficiency of UBE2T protein. Sanger sequencing of genomic DNA revealed a large paternal deletion and maternal duplication resulting from Alu-mediated recombination. In the absence of UBE2T, the patient cells are defective for FA pathway activation and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild type UBE2T demonstrating that deficiency of the protein UBE2T causes this individual’s FA. WES of a sibling pair with FA revealed biallelic mutations in FANCD1/BRCA2. Both siblings presented with multiple developmental abnormalities at birth, but did not develop any early childhood malignancies or hematological abnormalities typically associated with the FANCD1 complementation group. FANCD1/BRCA2 is best known for its role in homologous recombination directed repair of DNA double strand breaks, a function also required during the repair of ICLs. Each sibling inherited a LOF BRCA2 mutation in trans to a missense mutation of the BRCA2 DNA binding domain. Evaluation of BRCA2 DNA binding domain mutations revealed that this domain is important for replication fork protection, and to a lesser extent canonical homologous recombination. FA is a very heterogeneous disorder and as a consequence of overlapping clinical features, patients may be misdiagnosed with FA in lieu of another DNA repair or replication deficiency. Besides identifying FA mutations, we have identified non-FA patient enrolled in the IFAR. This individual has a defect in resolving DNA replication stress that presented in childhood as tri-lineage bone marrow failure, facial dysmorphia, and small stature. Our analysis demonstrated that the patient cells lack the hallmarks of FA, but are defective for cellular resistance to DNA replication stress