Diagnosis of fanconi anemia: chromosomal breakage analysis

Abstract

Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished so far. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. Cells derived from FA patients are-by definition-hypersensitive to DNA cross-linking agents, such as mitomycin C, diepoxybutane, or cisplatinum, which becomes manifest as excessive growth inhibition, cell cycle arrest, and chromosomal breakage upon cellular exposure to these drugs. Here we provide a detailed laboratory protocol for the accurate assessment of the FA diagnosis as based on mitomycin C-induced chromosomal breakage analysis in whole-blood cultures. The method also enables a quantitative estimate of the degree of mosaicism in the lymphocyte compartment of the patient.

Figure 1

Examples of chromosomal aberrations typically observed in a MMC-induced chromosomal breakage assay to diagnose FA. (a) Chromatid gap (broken piece in place); (b) chromatid break (broken piece dislocated); (c) chromatid interchange figure (“triradial”); (d) chromatid interchange figure (“quadriradial”); (e) other chromatid interchange figures. In the eventual analysis, (a) and (b) are counted as one, (c) and (d) as two break events. The left figure in (e) is counted as 8 break events (5 centromeres plus 3 open breaks); the right figure is equivalent to a quadriradial as in (d) (2 break events), in which two break points remained disconnected. (f), (g), and (h), are examples of nonconvincing “aberrations” that should be ignored in the analysis. (f) A gap that is not 100% convincing and should be ignored. (g) An association of 3 acrocentric chromosomes showing “satellite association”, not to be confused with a triradial, as in (c). (h) Two overlapping chromosomes, not to be confused with a true quadriradial, as in (d).

Figure 2

Evaluation of MMC-induced chromosomal breakage in stimulated T lymphocyte cultures. Upper row: healthy control; middle row: FA patient; lower row: mosaic FA patient. The healthy control shows breakage only at 300 nM, where the FA patient shows massive breakage (no normal cells present). Mosaicism is evident from the two highest concentrations of MMC, where there are still normal cells present next to cells showing an FA-like breakage rate (>10 breaks/cell). A crude estimate of the proportion of reverted T cells in this mosaic patient would be ~40%.