Abstract
[Purpose] We have studied the effects of A-bomb radiation on human germ cells, that is, whether the mutation rate has increased significantly in the offspring of A-bomb survivors compared with controls at DNA level. To conduct this study at genome-wide, we have been introducing a microarray based comparative genomic hybridization (array CGH) method. Before starting a large-scale study, we conducted a pilot study to examine the feasibility of this method.
[Experiments] We constructed an array with about 2400 Bac-clones. These clones are distributed about every 1.2 Mb across human autosomes. The results of preliminary studies demonstrated that our CGH system could reliably detected one copy change from normal duploidy. So, we have done a pilot study, in which we used genomic DNA from 40 offspring of A-bomb survivors and 40 members of a control group.
[Results and discussion] Variants observed once in this population, termed "rare variants", were identified on 14 Bac-DNA targets. The results from family studies revealed that these rare variants were inherited from at least one parent. Thus, until now, no de novo mutant was identified. We characterized these rare variants by Southern blot analysis using pulse field gel electrophoreses, FISH method, quantitative PCR, and sequencing. These results demonstrated that our array CGH system can reliably detect the variants larger than 50 kb. From our data, we consider that array CGH is one of the most useful techniques for our research purposes. Now, we are planning a larger population study.
