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Hypormorphic Fanconi anemia mutations
ABC
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Figure 1. Functional studies of the p.His913Pro and p.Arg951Gln variants. (A) Complementation analysis determined as cell survival after mitomycin C (MMC) treat- ment of lymphoblast (LFB) cells from patients F3 (homozygous for p.His913Pro) and F7 (compound heterozygous for p.Arg951Gln and p.Gly95Glufs*38) transduced with retroviral vector expressing the wild-type FANCA cDNA. (B and C) Complementation of the G2 cell cycle arrest after melphalan exposure of F3 and F7 LFB. (D and E) Comparison of the cell survival and cell cycle analysis in LFB cells of patients carrying the p.His913Pro (F3, F4, F5 and F6) and the p.Arg951Gln (F7) mutations and in control cells: wild-type (+/+) LFB cells and LFB cells not expressing the protein (-/-) due to a homozygous large intragenic deletion (c.284-?_1826+?del) of FANCA. There is no significant difference between LFB-/- cells and LFB carrying the missense mutations.
sion of intracellular reactive oxygen species (ROS) levels, and protection from proinflammatory cytokine-induced apoptosis.2 Indeed, FA cells have structurally abnormal mitochondria, as they appear swollen with matrix rarefac- tion, altered cristae and reticulum fragmentation.3,4 All these features further affect the mitochondrial functions, suppressing cell respiration, perpetuating ROS production and switching respiration from oxidative phosphorylation (OXPHOS) to aerobic glycolysis.2-5
An important milestone in unraveling the role of the FA proteins in the cytoplasm is the discovery of their involve- ment in selective autophagy.6 They are fundamental for removal of damaged mitochondria probably modulating mitochondrial fission-fusion balance, explaining why mutations of the FA genes result in accumulation of mor- phological defective mitochondria and inbalance of the cellular redox status.6,7
The clinical expressivity in FA is extremely variable from severe to mild, sometimes without clinical signs. Except for mutations of the FANCD2 and BRCA2 genes, no strong association has been found between the clinical phenotype with the genotype, mainly because of the wide genetic heterogeneity and the limited number of patients who belong to rare complementation groups.8,9 Even con- sidering FANCA, the most frequently mutated gene accounting for approximately two-thirds of the cases,10 the spectrum of mutation is extremly heterogeneous with pri- vate mutations and large intragenic deletions.11,12
We report functional studies of three missense FANCA mutations (p.His913Pro, p.Arg951Gln, and p.Arg951Gln), whose pathogenic effect in DNA repair is distinct from that in mitochondrial activity. The mutant proteins are
stably expressed but localized only in the cytoplasm, sug- gesting that they are functionally inactive at least for the DNA repair. They are instead functionally hypomorphic for the mitochondrial function. Consistent with the hypothesis of a residual activity of p.His913Pro, p.Arg951Gln, and p.Arg951Trp, the clinical phenotype of patients carrying these mutations is characterized by late onset of mild cytopenia, which tends to remain stable dur- ing follow up.
Methods
Biological samples
Eleven FA probands with positive chromosomal breakage test were included in this study. The institutional review board of the “G. Gaslini” Hospital, Genoa, Italy, approved the study, and all the subjects or their legal guardians gave written informed consent to the investigation according to the Declaration of Helsinki. In 10 cases (F2-F11), mutations had been previously reported.13 Patient P1 was a novel case analyzed by next generation sequencing.14
Complementation assay
Lymphoblast (LFB) cells were transduced with retroviral vectors expressing the cDNAs for FANCA, as previously reported.15,16 Mitomycin C (MMC) survival assay and cell cycle evaluation were performed as previously described.15
Western blot and immunofluorescence assay
A full-length FANCA sequence was amplified and cloned into the pcDNA3.1-Flag tagged expression vector. The mutant FANCA cDNAs (p.His913Pro and p.Arg951Gln/Trp) were generated by
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