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Hypormorphic Fanconi anemia mutations
minutes (min) in a proper medium and ATP synthesis was induced by the addition of 0.1 mM ADP. The reaction was mon- itored for 2 min, every 30 seconds (sec) in a luminometer (GloMax® 20/20n Luminometer, Promega Italia, Milan, Italy), by the luciferin/luciferase chemiluminescent method, with ATP stan- dard solutions between 10-8 and 10-5 M (luciferin/luciferase ATP bioluminescence assay kit CLSII, Roche, Basel, Switzerland). Data were expressed as nmol ATP produced/min/106 cells.
The oxidative phosphorylation efficiency (P/O ratio) was calcu- lated as the ratio between the concentration of the produced ATP and the amount of consumed oxygen in the presence of respiring substrate and ADP.21
Clinical data
Clinical information, phenotypic score and grade of cytope- nia was defined as described in Svahn et al.8 The hematologic condition was evaluated by scores grouped in three categories, from 1 to 3, from > 3 to 7, and > 7, representing mild, moderate, and severe phenotypes, respectively. Regarding the malforma- tion features, a score was assigned to every patient, dividing the population into three groups: mild (<6), moderate (6-15), and severe (>15).
Results
Missense p.His913Pro and p.Arg951Gln mutants of FANCA are stably expressed with loss-of-function effect on DNA repair
In our cohort of FA individuals, there are relatively recur- rent missense mutations of the FANCA gene, such as c.2738A>C (p.His913Pro) accounting for 9 alleles from six different families (Table 1). Three patients were homozy- gous (F1, F2, and F3) and another 3 (F4, F5 and F6) were compound heterozygous for the mutation with the second allele being a small in-frame p.Glu1239_Arg1243del or large intragenic deletion, and another missense p.Arg951Gln mutation, respectively. All the six families were from Sicily, suggesting a founder effect of the c.2738A>C variant. Indeed, genotyping four polymorphic loci closed to FANCA, we found that the variant was associated with the same haplotype in all the families (data not shown). Bioinformatics tools showed low pathogenicity scores for p.His913Pro.13 Consistently, the CADD score (score 13.25) is lower than the threshold (score 15.00) commonly used to predict pathogenicity of missense variants.22
AB
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 heterozy- gous (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 ##).
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