Diagnosis of Fanconi Anemia by Diepoxybutane Analysis

Abstract

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least 16 distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false-positive and falsenegative results. KeywordsFanconi anemia; DEB test; DNA interstrand crosslink repair; genomic instability Diagnosis of Fanconi anemia (FA) based on clinical manifestations alone is often difficult and unreliable because of the considerable overlap of the FA phenotype with that of a variety of genetic and nongenetic diseases (Table 8.7.1). It is currently considered standard of care that testing for crosslink sensitivity to rule out FA and testing for telomere length to rule out DKC should be part of the workup before treatment of pediatric acquired aplastic anemia (Williams et al., 2014).Although most FA patients exhibit abnormalities in growth parameters and skin pigmentation, approximately one third of the patients do not have any major birth defects (Giampietro et al., 1993(Giampietro et al., , 1997. Extensive genetic heterogeneity has been shown in FA patients, by use of various strategies for gene identification including functional and positional cloning, and more recently by mass spectrometric analysis of isolated protein complexes (Meetei et al., 2004), candidate gene sequencing in FA patient DNA (Kim et al., 2011), and by whole exome sequencing (Bogliolo et al., 2013). Sixteen unique genes (212) (Chandrasekharappa et al. 2013;Flynn et al., 2014), in order to individualize treatment for the patient, and provide genetic counseling regarding cancer risk for family members.FA affects approximately one in 100,000 live births. The average life expectancy is about 20 years, but some patients may not be diagnosed until the 3 rd decade of life or even later. Hematopoietic progenitor cells in FA are genetically unstable, leading eventually to bone marrow failure, which is the main life-threatening problem in these patients. This defect also predisposes to an increased frequency of clonal cytogenetic abnormalities, myelodysplastic syndrome (MDS), and acute leukemia. Development of any of these conditions is associated with poorer survival. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment currently for bone marrow failure in patients with...

Chromosome fragility in patients with Fanconi anaemia: diagnostic implications and clinical impact

“…Typically, reported indexes to measure chromosome fragility (‘breaks/cell’ or ‘percentage aberrant cells’) allow clear discrimination between the non-FA population and non-mosaic FA patients 17. However, mosaic patients present intermediate values and can be easily misdiagnosed.…”

“…Chromosome fragility tests on peripheral blood lymphocytes were performed basically as described by Auerbach,17 with some minor modifications. Three blood cultures were prepared for each patient, including 0.5 ml of blood in heparin and 4.5 ml of culture consisting of 15% fetal bovine serum, 1% antibiotics, 1% L-glutamine and 1% phytohaemagglutinin in RPMI (all reagents from Gibco, Carlsbad, CA, USA).…”

“…It was first observed in 1966, when Schroeder described a high frequency of spontaneously formed chromosome breaks in cells of patients with FA 13. Later, this genomic instability was seen to be highly induced by ICL-inducing agents, such as diepoxybutane (DEB), mitomycin C (MMC) or cis-platin,14 15 leading to the development of the first diagnostic test for FA 16 17. Although high sensitivity to ICL-inducing agents is the hallmark of FA cells, an accurate diagnosis is compromised in some cases, especially among mosaic patients, who represent 15–25% of all patients 18–20.…”

Fanconi Anemia Signaling and Cancer

“…At the cellular level, cells from FA patients are hypersensitive to DNA crosslinking agents and carry a distinct form of chromosomal abnormalities, which has been exploited as a clinical tool to diagnose FA [8, 9]. Together, the similar molecular features along with common clinical symptoms displayed from each FA sub-group suggest that the FA gene encoded products can function in one common signaling pathway.…”

Myelodysplasia and acute myeloid leukemia fifteen years after high-dose cyclophosphamide in a child with severe aplastic anemia

“… 1 Bone marrow aspirate (BMA) and biopsy examinations showed a hypoplastic specimen without megakaryocytes, with less than 15% cellularity and no dysplastic changes; cytogenetic studies were not available in our center at diagnosis. A test for hypersensitivity of Fanconi anemia (FA) cells to diepoxybutane, the diepoxybutane (DEB) test, which helps early diagnosis of FA 5 was not performed. The patient had no HLA-compatible donor and, at that time, therapy with ATG plus CS was not available in our country.…”