Determination of the degree of somatic mosaicism providing functional correction of Fanconi anemia (FA) hematopoiesis has direct implications for gene therapy for FA: it may help assess the percentage of FA hematopoietic cells corrected by gene therapy approaches that are needed to achieve clinically meaningful effects. Hypersensitivity to DNA interstrand cross-linking agents, such as diepoxybutane (DEB) and mitomycin C (MMC), is a cellular marker for diagnosis of FA. However, in some FA patients a population of DEB-resistant PHA-stimulated lymphoblasts (PHA-L) was observed, and this population sometimes varied over time. To assess the significance of this finding on hematopoietic function, we evaluated the MMC sensitivity of bone marrow mononuclear cells (BMMC) and DEB sensitivity of PHA-L and cultured lymphoblastoid cell lines (LCL) in 42 consecutive FA patients referred to the University of Minnesota. In cases where LCL were DEB-resistant, cultured fibroblasts were also studied. BMMC were cultured in the presence of increasing concentrations of MMC. PHA-L and LCL were cultured in DEB at 0.1 mcg/ml. Wild type BM progenitors (N = 17 subjects) proliferated regardless of increasing MMC concentrations (albeit at decreased efficiency at the highest concentrations) as follows: 0 MMC (normalized to 100%), 5 nM MMC (99% [standard deviation, SD, 16%]), 10 nM MMC (90% [SD 22%]), 25 nM MMC (77% [SD21%]), and 50 nM MMC (44% [SD 30%]). Of the 42 FA patients, BMMC failed to proliferate at 0 nM MMC in 10 patients and at 5 nM MMC in 20 patients. Twelve FA patients had MMC resistant BMMC: cells cultured in 5, 10, 25 and 50 nM MMC grew 44% (SD 28%), 35% (SD 24%), 24% (SD 30%) and 17% (SD 32%) of colony numbers in MMC free culture, respectively. Six of these 12 subjects were PHA-L mosaics as determined by DEB sensitivity testing. Four patients with no growth of BMMC at 0 or 5 nM MMC were also somatic mosaics in their PHA-L and LCL. Thus there was no clear correlation between somatic mosaicism as demonstrated by DEB testing in peripheral blood and sensitivity of BMMC to growth in MMC. Clinically, two patients with hematopoietic somatic mosaicism developed severe marrow aplasia, one of which received hematopoietic stem cell transplantation. Four of the mosaic patients had normal or near normal peripheral blood counts with one patient having clonal hematopoiesis by HUMARA assay and only low levels of metaphases with multiple breaks in multiple DEB studies. While patients with hematopoietic somatic mosaicism had mixed populations of DEB sensitive cells in their peripheral blood, all their fibroblast cultures were DEB sensitive. In summary, these data show that the presence of somatic mosaicism per se does not necessarily prevent bone marrow failure. Moreover, the data suggest that patients with stigmata of FA may have chromosomal breakage studies showing few cells (or no cells) with the characteristic changes of FA; in these cases, skin fibroblasts should be tested as well.

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