SCEs Are Markedly Elevated in Individuals with TOP3A Mutations
dHJ can be processed by two pathways; first, dissolution by the BTRR complex yields non-crossover products only; second, resolution by endonucleases that cleave Holliday junctions generate both non-crossovers and crossover products that are visualized as SCEs (Figure 3A). To determine if dHJ dissolution is impaired in the cells of affected individuals, we assessed the frequency of SCEs. After BrdU incorporation, we performed differential sister chromatid staining on primary fibroblasts and PHA-stimulated peripheral blood leukocytes (Figures 3B and 3C). Cells from individuals with TOP3A mutations had substantially (3- to 6-fold) more SCEs, than cells from control individuals or heterozygous parents (p < 0.0001 for all affected individual cell lines; Figure 3C). Therefore, excessive crossover recombination was evident in all tested individuals with TOP3A mutations, indicating that diagnostic cytogenetic assessment of SCE levels is predictive of TOP3A mutations as well as BLM mutations.
Figure 3 Individuals with TOP3A Mutations Have More SCEs, Reflecting Chromatid Hyper-recombination
(A) Schematic of dHJ processing. dHJs are either dissolved by the BTRR complex or alternatively cleaved by structure-specific nucleases that lead to dHJ resolution.
(B and C) BrdU strand-specific labeling of sister chromatids shows that TOP3A cells have more SCEs than control and parent cells. (B) Representative images of P1 and parent fibroblast cell lines. (C) Quantification of SCEs in fibroblast cell lines (P1, P5, P7, and P8) or PHA-stimulated lymphocytes derived from peripheral-blood samples (P2 and P3). The median value was plotted with more than ten metaphase spreads counted per subject. Pairwise non-parametric Mann Whitney tests were performed against parental control cells. F1, F2, and F5 SCEs were scored in independent laboratories.