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.
The aim of this work want to be a step towards the ongoing clarification of the molecular and cellular mechanisms that lead to genomic and chromosomal instability in such sites, associated with pathologic conditions.
Fanconi Anemia (FA) is the most representative type of chromosomal instability syndromes.
Analyze cells derived from a Fanconi Anemia patient is a great genetic model of the disease and particularly it is important to prove the strength of gene therapy for the correction of this pathological phenotype.
However, the most interesting feature of this approach is comparing the two isogenic cell lines of the same tissue: cells with mutated FANCA gene and cells with corrected FANCA gene from the same individual.
This allows to assess the differences related only with the defect in FANC A gene without the influences of genotype and tissue differences.
Moreover, this strategy has an optimal potentiality for constructing new approaches based on individual medicine, as a valid alternative of allogeneic Hematopoietic Stem Cell Transplantation (HSCT).
This current study converges on the high frequencies of chromosomal aberrations in FA cells and, in this optic, on using APH and DAPI treatments in order to better identify and understand the effects of their inductions in these cells. Although the relationship between APH-replicative stress in S phase and CFSs induction is known, post-replicative chromatin compaction during G2/M transition also appears to play equally a key role. Thus, use of DAPI could clarify how a different DNA conformation affects chromatin condensation and cellular processes.
Together these methodological approaches could fit a piece into the clinical picture of Fanconi Anemia and, hopefully, could lay a more solid basis for a finer preventive and therapeutic resolution of the disease symptoms and cancer susceptibility.