Diagnosis of Fanconi anemia in patients with bone marrow failure

Abstract

Background: Patients with bone marrow failure and undiagnosed underlying Fanconi anemia may experience major toxicity if given standard-dose conditioning regimens for hematopoietic stem cell transplant. Due to clinical variability and/or potential emergence of genetic reversion with hematopoietic somatic mosaicism, a straightforward Fanconi anemia diagnosis can be difficult to make, and diagnostic strategies combining different assays in addition to classical breakage tests in blood may be needed.

Design and methods: We evaluated Fanconi anemia diagnosis on blood lymphocytes and skin fibroblasts from a cohort of 87 bone marrow failure patients (55 children and 32 adults) with no obvious full clinical picture of Fanconi anemia, by performing a combination of chromosomal breakage tests, FANCD2-monoubiquitination assays, a new flow cytometry-based mitomycin C sensitivity test in fibroblasts, and, when Fanconi anemia was diagnosed, complementation group and mutation analyses. The mitomycin C sensitivity test in fibroblasts was validated on control Fanconi anemia and non-Fanconi anemia samples, including other chromosomal instability disorders.

Results: When this diagnosis strategy was applied to the cohort of bone marrow failure patients, 7 Fanconi anemia patients were found (3 children and 4 adults). Classical chromosomal breakage tests in blood detected 4, but analyses on fibroblasts were necessary to diagnose 3 more patients with hematopoietic somatic mosaicism. Importantly, Fanconi anemia was excluded in all the other patients who were fully evaluated.

Conclusions: In this large cohort of patients with bone marrow failure our results confirmed that when any clinical/biological suspicion of Fanconi anemia remains after chromosome breakage tests in blood, based on physical examination, history or inconclusive results, then further evaluation including fibroblast analysis should be made. For that purpose, the flow-based mitomycin C sensitivity test here described proved to be a reliable alternative method to evaluate Fanconi anemia phenotype in fibroblasts. This global strategy allowed early and accurate confirmation or rejection of Fanconi anemia diagnosis with immediate clinical impact for those who underwent hematopoietic stem cell transplant.

Figure 1.

A new flow-based MMC-sensitivity test to diagnose Fanconi anemia (FA) on fibroblasts. (A) Graphs representing the MMC-sensitivity data in FA and non-FA cells: on the X axis are plotted the minimal MMC concentration at which dying cells were detected; Y axis are the number of cases. FA (top panel) and non-FA (middle panel) controls, including other fragility syndromes, and BMF patients (bottom panel) data are shown. (B) MMC-sensitivity flow data from two NBS1-mutated Nijmegen patients fibroblasts, compared to a non-FA and a FA-A (EGF125) control patients. FA-A, but not the NBS1-mutated fibroblasts, clearly demonstrated greater sensitivity to MMC compared to non-FA control in the same experiment, as detected by PI intracellular uptake.

Figure 2.

Flow-chart of the Fanconi anemia (FA) work-up in the 87 BMF patients. PBL and primary fibroblasts were tested as indicated. Skin fibroblasts were obtained in 64 patients. For breakage tests, negative (n=75) and failure (n=7, no metaphase obtained) cases were grouped to make the figure simpler, considering that all failure cases further turned out to be non-FA (normal FANCD2 pattern in PBL and normal results in fibroblasts). In the FA patients who were diagnosed, 5 were further grouped as FA-A, one was FA-C, and one was ‘downstream’ with hematopoietic mosaicism. FANCA and FANC mutations were identified, and complete FANCA reversion was found in one allele in the PBL of the 2 FA-A with somatic mosaicism (Figure 3). Abbreviations: FA core, no FANCD2-L isoform; MMC-S, hypersensitivity to mitomycin C (MMC); MMC-R, no hypersensitivity to MMC.

Figure 3.

FA diagnosis in patient H04 with hematopoietic somatic mosaicism. (A) Western blot demonstrated an FA core pattern in the primary fibroblasts (no FANCD2-L isoform, indicated by a star) but a normal pattern in PBL (arrow), respectively. Control FA and non-FA samples are also shown, including an FA-D2 sample in line 3 (with no detectable FANCD2 protein; FANCD2 mutations were detected in this patient, not sho ). ( B)wn MMC sensitivity test demonstrated a clear hypersensitivity in patient H04/EGF056 (line 2) compared to non-FA (line 1) and FA-A EGF008 (line 3) controls. Arrows show dying cells at lower MMC concentration for each patient as detected by PI uptake. (C) Two FANCA mutations were identified in the fibroblast DNA; Mutation c.[1115-1118del4]/p.[V372A fs X42] (exon 13, arrow) was found in the fibroblast gDNA (top panel) but not in the PBL gDNA (lower panel), demonstrating complete somatic mosaicism.

Figure 4.

Suggested diagnostic strategy for evaluating Fanconi anemia in BMF. Initial assessment should include thorough clinical evaluation with detailed personal/family history and physical exam, and chromosomal breakage test in PBL. Patients with no positive FA signs and no increased breaks are considered to be non-FA and are treated accordingly. If breaks are increased, a diagnosis of FA is made and appropriate treatment/follow-up offered. Primary skin fibroblast analyses should be considered in patients where a suspicion of FA remains after negative chromosomal breakage test on PBL (based on positive findings in the clinical assessment), or in patients with inconclusive tests in blood.