Research Article: Diagnosis of Fanconi Anemia: Chromosomal Breakage Analysis

Date Published: May 24, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Anneke B. Oostra, Aggie W. M. Nieuwint, Hans Joenje, Johan P. de Winter.


Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished so far. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. Cells derived from FA patients are—by definition—hypersensitive to DNA cross-linking agents, such as mitomycin C, diepoxybutane, or cisplatinum, which becomes manifest as excessive growth inhibition, cell cycle arrest, and chromosomal breakage upon cellular exposure to these drugs. Here we provide a detailed laboratory protocol for the accurate assessment of the FA diagnosis as based on mitomycin C-induced chromosomal breakage analysis in whole-blood cultures. The method also enables a quantitative estimate of the degree of mosaicism in the lymphocyte compartment of the patient.

Partial Text

Fanconi anemia (FA) is a cancer-prone chromosomal instability disorder with diverse clinical symptoms (Table 1) [1]. Because of its rarity and variable presentation FA may be heavily underdiagnosed [2, 3]. Clinical suspicion of FA is mostly based on growth retardation and congenital defects in combination with life-threatening bone marrow failure (thrombocytopenia and later pancytopenia), which usually starts between 5 and 10 years of age. However, the clinical manifestations are highly variable, while some of the symptoms may overlap with those observed in other syndromes, making a reliable diagnosis on the basis of clinical features virtually impossible (Table 1). Even patients presenting with a number of “typical” FA symptoms may not be suffering from FA. Cells derived from true FA patients must exhibit a hypersensitivity to chromosomal breakage induced by DNA cross-linking agents such as mitomycin C (MMC), diepoxybutane (DEB), or cisplatinum.

Here we describe a laboratory protocol that has evolved during 30 years of experience and which we can recommend for the unambiguous diagnosis of the vast majority of FA patients, including patients with hematopoietic mosaicism. The test is based on the 72 hour whole-blood cultures as routinely applied in cytogenetics laboratories to make chromosomal preparations for karyotypic analysis. Metaphase spreads are Giemsa-stained (not banded) and analyzed for microscopically visible chromatid-type aberrations. For technical details the reader is referred to the appendices. Laboratories that are not set up to do this type of assay or have no experience with diagnosing FA on a regular basis should be advised to refer to a laboratory where the test is applied on a routine basis, rather than attempting to carry out a “similar” test that is considered a plausible alternative. The test might be omitted if a proband belongs to an ethnic population with a high carrier frequency for a specific FA gene mutation. Demonstrating this mutation in the proband would be diagnostic for FA, although the result may not provide information about possible mosaicism.

It should be pointed out that, even though we have chosen to use MMC as the cross-linking agent, DEB is used in a widely accepted alternative protocol [1, 26–28]. Pros and cons for using the various cross-linking agents are further discussed in the appendices.