The application of spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) technology to determine the chromosomal content(s) of micronuclei

Abstract

DNA loss by the process of micronucleation is associated with aging, cancer and environmental exposure. The primary aim of this study was to identify the chromosomal origin of the DNA excluded into micronuclei (MN). This was achieved using a novel application of SKY and FISH technologies. Cytochalasin B (Cyt B)-treated lymphocyte cultures from three females (aged 28, 42 and 72) were analyzed. SKY revealed that the majority of MN (89.8, 82.9, and 97.6% in the 28-, 42- and 72-year-old (y.o.), respectively) had a uniform, single color, suggesting that they were comprised of DNA from a single chromosome. Using a pancentromeric probe, most of the MN (82% in 28 y.o., 69% in 42 y.o. and 80% in 72 y.o.) had one centromere signal present. Overall, the confirmation studies (using FISH with chromosome-specific WCP) were in agreement with the SKY chromosomal assignments for 71.1% of the MN. Although the SKY analysis showed that all of the 23 chromosomes (22 autosomes and the X chromosome) could be present in the MN, overall, the X chromosome was seen most frequently. DNA from the X chromosome was seen in 50.6% of MN in the 42 y.o. individual, whereas in the 28 and 72 y.o. it was seen in 12.2 and 7.1% of MN, respectively. This difference ( P<0.0001) in the frequencies of X chromosome exclusion into MN among individuals was independently confirmed using a single whole chromosome painting probe (WCP) for the X chromosome. SKY also showed variation in the frequency of autosomal exclusion into MN between chromosomes and between females. Collectively, this study supports the hypothesis that the majority of MN contain DNA from a single, monocentric chromosome. The use of SKY technology for the identification of the chromosomal content(s) of MN provides an opportunity for expansion of our knowledge of the chromosomal changes that accompany MN formation.