Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia

Abstract

Fanconi anemia is a rare disease characterized by congenital malformations, aplastic anemia, and predisposition to cancer. Despite the consolidated role of the Fanconi anemia proteins in DNA repair, their involvement in mitochondrial function is emerging. The purpose of this work was to assess whether the mitochondrial phenotype, independent of genomic integrity, could correlate with patient phenotype. We evaluated mitochondrial and clinical features of 11 affected individuals homozygous or compound heterozygous for p.His913Pro and p.Arg951Gln/Trp, the two residues of FANCA that are more frequently affected in our cohort of patients. Although p.His913Pro and p.Arg951Gln proteins are stably expressed in cytoplasm, they are unable to migrate in the nucleus, preventing cells from repairing DNA. In these cells, the electron transfer between respiring complex I-III is reduced and the ATP/AMP ratio is impaired with defective ATP production and AMP accumulation. These activities are intermediate between those observed in wild-type and FANCA-/- cells, suggesting that the variants at residues His913 and Arg951 are hypomorphic mutations. Consistent with these findings, the clinical phenotype of most of the patients carrying these mutations is mild. These data further support the recent finding that the Fanconi anemia proteins play a role in mitochondria, and open up possibilities for genotype/phenotype studies based on novel mitochondrial criteria.

Figure 1.

Functional studies of the p.His913Pro and p.Arg951Gln variants. (A) Complementation analysis determined as cell survival after mitomycin C (MMC) treatment 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.

Figure 2.

Expression of p.His913Pro and p.Arg951Gln FANCA proteins. (A) Western blot of total lysates from lymphoblast (LFB) cells of patients carrying the p.His913Pro (F3, F4, F5 and F6) and the p.Arg951Gln (F7 and F10) mutations, showing that the mutant FANCA proteins are expressed. The controls are wild-type (+/+) LFB cells and LFB cells not expressing the FANCA protein (−/−) due to a homozygous large intragenic deletion (c.284-?_1826+?del) of FANCA. (B) Western blot of cytoplasmic (C) and nuclear (N) fractionated cellular lysates after 2 mM hydroxyurea treatment (24 hours) of LFB from patients F3 and F7, showing that the endogenous p.His913Pro and p.Arg951Gln proteins do not translocate to the nucleus. Controls +/+ and −/−, as indicated in (A). (C) Western blot of cytoplasmic (C), nuclear (N) fractionated, or total (TL) cellular lysates from 293T cells transfected with the wild-type (wt) or the mutant (H913P and R951Q) forms of FANCA tagged with FLAG, confirming the exogenous p.His913Pro and p.Arg951Gln FANCA proteins are retained in the cytoplasm. (D) Immunofluorescence analyses on 293T cells transfected as indicated in (C). Nuclei are stained with propidium iodide (PI). (E) Western blot of different LFB cells exposed to 2 mM hydroxyurea (24 hours) showing no monoubiquitination of the FANCD2 protein. Control +/+, as indicated in (A).

Figure 3.

Hypomorphic effect of missense mutations on mitochondrial activity. (A) Intracellular concentrations of ATP and AMP, ATP/AMP ratio, and electron transfer between complex I and III were determined in lymphoblasts (LFB) from patients with mild (F6, F7), moderate (F3, F4) and severe (F6) hematologic scores. LFB from 3 Fanconi anemia (FA) patients not expressing FANCA (−/−) and 5 healthy individuals (+/+) have been used as controls. The three −/− cell lines are compound heterozygous (p.Arg18Profs*19/p.Asn1221Thrfs*26 and p.Ser175Leufs*5/p.Trp183*) and homozygous (Gly95Glufs*31) for nonsense or frameshift mutations. Each graph is representative of three experiments carried out in each cell line and data are expressed as mean±Standard Deviation (SD). t-test indicates a significant difference of P<0.05 (*) and P<0.01 (**) between the +/+ cells and the other samples, while # and ## indicate a significant difference of P<0.05 and P<0.01, respectively, between the −/− LFB cells and the other samples. (B) The same parameters reported in (A) were evaluated in an LFB −/− cell line transfected with the wild-type (wt) or mutant (H913P, R951Q and R951W) forms of FANCA tagged with FLAG, showing that the mitochondrial activity is intermediate between that of LFB +/+ and LFB −/−cells transfected or untransfected (+/+ or −/− vectors) with the empty vector. Each graph is representative of three experiments carried out in each cell line and data are expressed as mean±Standard Deviation (SD). t-test indicates a significant difference of P<0.05 (* or #) and P<0.01 (** or ##) between LFB cells −/− transfected with empty vector or expressing the three missense mutant forms of FANCA and LFB cells +/+ (* and **) or LFB cells −/− (# and ##).

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 −/−.