Fanconi Anemia and Chromosome Instability
Topic: Fanconi Anemia (FA) is a polygenic inherited syndrome caused by DNA repair deficiency in one of the 21 FANC genes. Around the 60-70% of clinical cases present mutations in FANC A gene.
Fanconi anemia is characterized by chromosomal instability and a prolonged G2 phase of the cell cycle to rescue DNA lesions accumulated during an impaired replication process in S phase.
Given the impossibility to repair ICLs (Interstrand DNA Crosslinks) and UFBs (Ultrafine Anaphase Bridges), FA cells show high percentages of chromosomal aberrations in metaphases spreads.
The sites mainly involved in chromosomal aberrations in FA cells are Common Fragile Sites (CFSs). These genomic sites are late/delayed replicating regions and are expressed as chromosome gaps and/or breaks.
Purpose: analyze the most expressed CFSs in two isogenic lymphoblastoid cell lines, one mutated in the FANC A gene (HSC72 FA-A) and the other one transfected with normal FANC A gene (HSC72 FANCA). Furthermore, the investigation of replication timing within CFSs in control and under different stress conditions (APH and DAPI) can reveal how the replication process can contribute to genomic instability in FA lymphoblasts.
Finally, it is possible to recognize the implication of FA repair pathway in ensuring chromosome integrity, comparing the two different molecular and clinical backgrounds with peripheral blood lymphocytes from healthy individuals.
Methods: Giemsa and Chromomycin A3 staining can evidence the chromosomal aberrations and FISH (Fluorescent In Situ Hybridization) can map CFSs on metaphases. The combination of FISH and IF (ImmunoFluorescence) against BrdU (BromodeoxyUridine) and against H2AXp (phosphorylated Histone 2AX) could identify each S-phase substages/G2 phase and damaged CFSs, respectively. Lastly, RNA-FISH technique allows recognition of the RNA:DNA hybrids, generated by the simultaneous spatial and temporal collision of replication and transcription processes within large genes at CFSs.