Fanconi anemia (FA) is a cancer-prone chromosomal instability disorder with diverse clinical symptoms () [1
]. Because of its rarity and variable presentation FA may be heavily underdiagnosed [2
]. 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 (). 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.
General features and symptoms associated with Fanconi anemia.
Indications to test for FA are typical congenital abnormalities with/without thrombocytopenia and/or marrow failure. However, congenital abnormalities may be absent, while isolated thrombocytopenia may be the only presenting symptom. In all children with aplastic anemia FA should be tested as the possible underlying disease. In children and adults with cancer and an unusual response to DNA-damaging agents such as chemotherapy or radiotherapy (severe skin reactions or mucositis, longlasting aplasia), FA should also be tested for. Similarly, in adults with carcinomas (typically located in the mouth/esophagus or anogenital region) at relatively young age, FA should be considered. Cancer or leukemia may be the first symptom of FA, while congenital abnormalities and marrow failure may be absent altogether, the latter especially in cases with hematopoietic mosaicism [4
The cellular phenotype typical for FA is ascertained using phytohaemagglutinin-stimulated whole-blood (T lymphocyte) cultures. Although it has been considered the gold standard for diagnosing FA, the test is not 100% specific. A few cases of Nijmegen breakage syndrome have been reported to give a false positive result [7
], which can be excluded by screening the NBS1
gene for mutations. In addition, patients suffering from the cohesinopathies Roberts syndrome (mutated in ESCO2
) and Warsaw breakage syndrome (mutated in DDX11
) may score positive in the test [10
]. Additional “atypical FA” or “FA-like” patients have been reported as case reports [11
]. Somewhat controversially, the “FA-like” patient found to be mutated in RAD51C
has been assigned to a distinct genetic FA subtype (FA-O) [13
Approximately 80% of the patients referred for FA diagnostic testing because of bone marrow failure score negative in the chromosomal breakage test. These “true negatives” have other causes of marrow failure and most often represent cases with acquired aplastic anemia.
Lymphocyte mosaicism occurs in a sizable proportion of FA patients (estimated at 10–30%) and is caused by spontaneous genetic reversion at the disease locus in hematopoietic progenitor cells; the reverted cells may (partially) correct the bone marrow failure [14
]. In most of these cases FA can still be diagnosed by testing peripheral blood, since a portion of the cells will still show hypersensitivity to cross-linking agents. Occasionally, the percentage of reverted cells has reached such a high level as to produce a false negative diagnosis. In such cases cross-linker sensitivity may be tested in skin fibroblasts, which are not known to be affected by mosaicism. After a positive breakage test result has been obtained, screening for mutations in the known FA genes is warranted.
Laboratory studies have revealed as many as 15 distinct “complementation groups” or genetic subtypes: FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, and -P [13
]. For all subtypes known to date the disease genes have been identified. Global relative prevalences are difficult to estimate, as the values may differ considerably depending on the ethnic background, due to founder effects. All FA genes are localized on autosomes, except FANCB
, which is X-linked and subject to X inactivation in female carriers [22
]. These two different modes of inheritance have important consequences for the counseling of FA families.
Recognition of FA as a chromosomal instability disorder was originally based on chromatid-type aberrations spontaneously occurring in standard cytogenetic preparations. However, this phenomenon was later found to be highly variable and considered not reliable for diagnostic purposes. After the discovery of an extreme sensitivity of FA cells to the chromosome-breaking effect of the cross-linking agents mitomycin C (MMC) [23
] and diepoxybutane (DEB) [24
], this feature has become routinely utilized to diagnose FA by a “chromosomal breakage test.” In this test, T lymphocytes in a peripheral blood sample are cultured in the presence of a cross-linking agent, after which chromosomal aberrations are quantified in metaphase spreads. Numerous variations of the test are used in the various cytogenetic laboratories, with significant differences in exposure times and drug concentrations. Also, the ways in which data are evaluated are diverse. We have encountered opposite conclusions from different laboratories based on the very same primary data set, due to a lack of experience in performing the test and evaluating the resulting data. Evidently, there is a great need for a clearly described reliable protocol for the accurate diagnosis of FA patients.