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R. Bottega et al.
To exclude the possibility that another FA gene or another unknown FANCA variant in linkage disequilibri- um with c.2738A>C could be responsible for the disease, we further investigated the role of FANCA as a disease- causing gene. First of all, complementation analysis of FANCA restored the MMC sensitivity and/or G2 arrest
induced by melphalan in LFB homozygous for p.His913Pro (F3) (Figure 1A and B). Then, to investigate the functional impact of p.His913Pro on protein stability and localization, we performed Western blot analysis using LFB from 4 affected individuals (F3-F6). In all the four cell lines, FANCA was not degraded and was
A
B
Figure 4. Oxymetric measures and ATP synthesis. (A) Amperometric traces of the oxygen consumption for LFB FANCA-/- cells transfected with wild-type (+/+), p.His913Pro, p.Arg951Gln, and p.Arg951Trp mutant forms of FANCA. (B) ATP synthesis rate in the same samples as in (A). The data in (A) and (B) are also depicted as histograms. Each bar graph is representative of three experiments and data are expressed as mean±Standard Deviation (SD). Anova test indicates a significant difference of P<0.05 (*) and P<0.01 (**) between the +/+ sample and the other samples, while ## indicates a significant difference of P<0.01 between LFB -/- transfected with the p.His913Pro, p.Arg951Gln and p.Arg951Trp mutant forms of FANCA and LFB -/-.
haematologica | 2018; 103(3)