Page 64 - Haematologica3

R. Bottega et al.
Patients F5 and F8 were the 2 affected individuals with severe hematologic scores. In addition to having multiple congenital malformations (score 22), F5 was characterized by severe tri-lineage requiring blood transfusions. He suc- cessfully underwent HSCT two years after diagnosis. At diagnosis, F8 had no congenital malformation except for growth delay and mild thrombocytopenia. However, the mono-lineage cytopenia evolved in severe tri-lineage cytopenia concomitantly with myelodysplastic syndrome transformed in acute myelogenous leukemia. He died three years later for relapse after two HSCT.
Discussion
Mutations of the FA genes are almost private, though common founder mutations have been reported in few populations, including Spanish Gypsies, the Afrikaner population of South Africa, Ashkenazi Jews, and people from some Italian geographical regions.24-27 This is the case of p.His913Pro, a mutation of the FANCA gene that is rel- atively frequent, even as a homozygous condition, in patients from Sicily. Indeed, the analysis of microsatellite markers in the six families revealed the presence of a com- mon haplotype compatible with a founder effect. In con- trast, p.Arg951Gln has been identified, never as a homozygous mutation, in individuals from different geo- graphical areas, suggesting that it occurred as independent de novo mutational events. At the same residue, an addi- tional rare pathogenic mutant (p.Arg951Trp) affects one allele of our cohort of patients.23
Since these alterations are missense mutations, their pathogenic effect on protein function has been ascertained by considering several aspects. Unlike p.Arg951Gln/Trp, p.His913Pro was classified as a variant of uncertain signif- icance, as the predictive bioinformatic tools indicated that this amino acid substitution has no or mild effect on pro- tein function.13 However, genetic studies support their pathogenic role, as they are present in FA patients and only rarely in controls (ExAC or other databases).13 Consistent with this hypothesis, we found that the mutant FANCA proteins (both endogenous and over- expressed) localize only in the cytoplasm, where they are stably expressed at similar levels to those observed in wild-type cells. As a consequence, the FA/BRCA pathway remains inactive because FANCD2 is not monoubiquiti- nated, as demonstrated by Western blot analysis.
These data are consistent with those reported by Castella et al.,24 who showed that all the FANCA missense mutations are stably expressed. Like p.His913Pro and p.Arg951Gln, their products do not enter the nucleus, pre- venting FANCD2 being monoubiquitinated. On the con- trary, the same authors demonstrated that null mutations, such as nonsense or frameshift alterations, are unstable and not detectable within cells.24 Therefore, our data con- firm the hypothesis of a correlation between type of mutation and expression level of mutant FANCA, but as to why these mutant proteins are retained in the cytoplasm without any apparent function remains a subject of debate.
In trying to unravel any role of the FA proteins in other cell compartments, we investigated the mitochondria OXPHOS function and the cellular energy status whose alteration may lead to an impaired mitophagy process.28 As demonstrated by biochemical assays, expression of the
p.His913Pro and p.Arg951Gln/Trp proteins are associated with a mild mitochondrial function impairment. In partic- ular, the electron transport between complexes I and III, the ATP production, and the O2 consumption appear defective but not to the same extent as in FA cells homozygous for null mutations. Interestingly, the defec- tive OXPHOS is limited to the pathway composed by complexes I, III and IV, considering that the oxygen con- sumption and the relative ATP synthesis is similar in con- trol and FA samples when induced by succinate. This sug- gests that the potential function of FANCA, as well as other FA proteins, on mitochondria is confined to the OXPHOS led by complex I. However, the evaluation of P/O ratio shows that, in all samples, the oxygen consump- tion, even when very low, is finalized to the ATP synthe- sis, indicating that the mitochondria are always in a cou- pled status.
In accordance with the recent discovery that the FA pro- teins are required for clearance of damaged mitochondria and to decrease the mitochondrial reactive oxygen species,6 our finding supports the role of FANCA in these organelles. Considering that small interfering RNA of the FA genes is associated with increased puncta, which is a marker for defects in clearance of damaged mitochondria,6 we can speculate that mutations of FANCA could nega- tively influence the efficiency of the mitophagy, causing an accumulation of damaged mitochondria. This may determine not only a decrement in the energy production, but also the increment of oxidative stress, which could induce damage of other mitochondria. However, we can- not exclude the possibility that the FANCA protein may have a more direct role in the modulation of the mito- chondria structure and function, i.e. influencing the organ- ization of inner mitochondrial membrane, whose integrity is essential for the proper functioning of the OXPHOS.
Of interest, our data allow us to dissect the activity of FANCA in DNA repair from that in preserving the mito- chondrial function, as has previously been hypothesized for FANCC.6 Indeed, one mutation of this gene (c.67delG), resulting in an N-terminus truncated but stable mutant form of FANCC, is unable to repair DNA but is able to restore the mitochondrial function. Therefore, we regard the p.His913Pro and p.Arg951Gln/Trp as variants with a hypomorphic effect of FANCA in maintaining the mito- chondrial activity. Even mutations of the FANCD2 gene have been regarded as hypomorphic.29 However, their 'hypomorphic' effect is associated with the role of the protein in controlling genomic integrity. In FANCD2 indi- viduals, at least one allele is always associated with expression of a low amount of FANCD2, which is detectable in both the non- and monoubiquitinated form, suggesting that residual activity of FANCD2 for the DNA repair processes is essential for life. On the contrary, bial- lelic null mutations of FANCA and FANCC are relatively common in FA and are always associated with lack of FANCD2 monoubiquitination.24
Considering that the c.67delG mutation of FANCC, which is able to restore the mitochondrial function,6 is associated with mild clinical phenotypes,9,30 we evaluated whether we could reach the same conclusion in our cohort of 11 patients. The mean age (8.9 years) at diagno- sis was higher than that reported in the FA Italian cohort (6.8 years).8 Nine of them had concordant mild/moderate hematologic and somatic phenotypes. Regarding malfor- mations, 4, 6, and one patients were classified as mild,
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haematologica | 2018; 103(3)