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Hypormorphic Fanconi anemia mutations
expressed at similar level as in wild-type cells (FANCA+/+) (Figure 2A). The protein was instead unde- tectable, even at different exposures, in LFB (FANCA-/-) from an FA patient homozygous for a FANCA large intra- genic deletion (exons 4 to 20) resulting in a frameshift mutation (p.Gly95Glyfs*31). Finally, in order to evaluate whether the mutant p.His913Pro protein could play a role in nuclear DNA repair, we analyzed fractionated cellular lysates. As expected, FANCA was detected in both cyto- plasm and nucleus of the wild-type LFB cells. Instead, the protein was present only in the cytoplasm of cells from F3 (Figure 2B). Immunofluorescence and Western blot of frac- tionated cellular lysate from the 293T cells over-express- ing the wild-type or the mutated FANCA tagged with FLAG confirmed that the mutant protein was retained in the cytoplasm (Figure 2C and D). Taken together, these data indicated that the p.His913Pro mutant form of FANCA is stably expressed but is unable to enter the nucleus and play its role in the DNA repair pathway.
Of note, patient F6 is a compound heterozygous for the p.His913Pro and p.Arg951Gln mutations. The latter was identified in another 4 probands (F7, F8, F9, and F10) who are compound heterozygous for deleterious frameshift, splicing or start-loss mutation of FANCA (Table 1). Consistent with haplotype analysis (data not shown), the families carrying p.Arg951Gln come from different geo- graphical areas, suggesting that the mutation originated from independent de novo events and that the presence of another disease-causing mutation in linkage disequilibri- um was unlikely. In the LFB cells of F7, the sensitivity to MMC and the G2 arrest were restored by complementa- tion analysis (Figure 1A and C). Like the p.His913Pro sub- stitution, p.Arg951Gln did not affect FANCA stability, being the protein expressed in LFB of patients F7 and F10 (Figure 2A). Moreover, neither the endogenous nor the over-expressed mutant protein migrated into the nucleus (Figure 2B and C).
Taken together, these data suggest that p.His913Pro and p.Arg951Gln are loss-of-function mutations for the role of FANCA in controlling the genomic integrity. Consistent with this hypothesis, FANCD2 was not monoubiquitinat- ed in any of the six cell lines available for the analysis (Figure 2E). Moreover, in the same cells, the sensitivity to MMC and the percentage of cells blocked in the G2 phase are comparable with those of the FANCA-/- cells (Figure 1D and E).
Finally, at position 951 an additional substitution (c.2851C>T/p.Arg951Trp) was identified in one allele of patient F11. However, the LFB cell line or other biological samples were not available for investigations to confirm that the mutation is associated with protein stability and lack of FANCD2 monoubiquitination, as reported by Karras et al.23
p.His913Pro and p.Arg951Gln are hypomorphic mutations for mitochondrial activity
In order to evaluate whether FANCA could affect the mitochondrial function, we investigated the electron transport chain functionality and the cellular energy status in LFB from patients with mild (F6 and F7), moderate (F3 and F4), and severe (F5, the only LFB cell line available within this group) hematologic score. Consistent with the FA mitochondrial phenotype,2-4 the cells showed an altered ATP/AMP ratio with reduced ATP production and AMP accumulation, and impaired electron transfer between
complexes I and III (Figure 3A). Of note, the biochemical values were intermediate between those obtained in FANCA+/+ and FANCA-/- cells, suggesting that p.His913Pro and p.Arg951Gln are hypomorphic alleles for the mitochondrial activity.
In order to demonstrate that these alleles are directly involved in determining a mild mitochondrial phenotype, we over-expressed the p.His913Pro and p.Arg951Gln, as well as p.Arg951Trp, FANCA proteins in FANCA-/- cells. Whereas the ATP and AMP concentrations, the ATP/AMP ratio, and the electron transport were complemented by transfection of the wild-type cDNA, the same phenotypes were only partially restored by expression of the three mutant cDNAs. Of note, the biochemical measurements are significantly different when FANCA-/- cells are com- pared with those over-expressing the mutant forms of FANCA (Figure 3B).
The defect in the electron transfer between complexes I and III determines an impaired oxygen consumption and ATP synthesis in FANCA-/- cells, while in the samples expressing His913Pro, Arg951Gln and Arg951Trp, this activity is partially recovered, although with values lower than those obtained in cells expressing the wild-type FANCA protein (Figure 4A and B). Conversely, respiration was observed in all samples after stimulation with succi- nate, indicating that the electron transfer through complex II, III and IV pathways is not impaired (Figure 4A). This implies that the FA mutations may have a specific interac- tion with the complexes I, III and IV pathway. However, similarly to that reported in literature,21 the P/O ratio in the presence of pyruvate/malate is around 2.5 in all sam- ples, suggesting that, despite the impaired mitochondrial function in FANCA-/- and in cells expressing the three mutant forms of FANCA, the residual oxygen consump- tion is devoted to ATP synthesis (Figure 4C).
Moderate clinical phenotype in patients with missense mutations affecting residues His913Pro and Arg951
The clinical data of the affected individuals carrying the p.His913Pro, p.Arg951Gln, and p.Arg951Trp mutations are reported in Table 1. Of the 11 patients, 8 were male and 3 females. Mean age at diagnosis was 8.9 years (range 4-16 years). Ten had mild or moderate somatic phenotype (range 1-11). Nine of these individuals had concordant mild or moderate hematologic scores ranging from 2 to 8. Three individuals (F1, F6, and F7) had mild cytopenia that did not require transfusions. Of note, F6 and F7 had a mild leukopenia/neutropenia at diagnosis; at present, blood count is in the normal range in F6 and neutropenia is sta- ble in F7 after 4 and 11 years, respectively, from the first abnormal blood count. The remaining 6 probands under- went HSCT. The first abnormal blood count was a mild tri-lineage cytopenia in F2 and F9 or thrombocytopenia, as in F10 and F11. They all underwent HSCT for progression to aplastic anemia, which occurred 10-11 years after diag- nosis in F2, F9, and F11. Even F3 and F4 did receive HSCT after two and five years, respectively, of stable moderate cytopenia because a matched healthy sibling brother was available. Except for F9, who died for HSCT complica- tions due to cytomegalovirus infection, the other patients are all alive after a follow up ranging from 1 to 18 years (mean 9.5 years) and none have developed hematologic adverse events (myelodysplastic syndrome, acute myel- ogenous leukemia or cytogenetic alterations) and/or solid tumor.
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