Diagnosis of fanconi anemia: chromosomal breakage analysis.

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.

1. Introduction

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.

Table 1

General features and symptoms associated with Fanconi anemia.

Birth prevalence	0.5–2.5 per 105 newborns; varies with ethnic background.
Mode of inheritance	Autosomal recessive (>98%) and X-linked (~1-2%).
Carrier frequency	Traditional overall estimate: “1/300 worldwide.” Needs reassessment according to subtype and ethnic background.
Congenital abnormalities*	Radial ray abnormalities (aplastic or hypoplastic radii and absent or extra thumbs) and other skeletal abnormalities; small head circumference; abnormal shape of the ears; microphthalmia; ectopic or horse-shoe kidney; hypogonadism; heart abnormalities; intestinal or anal atresia.
Other somatic abnormalities*	Short stature/retarded growth; reduced fertility; skin pigmentation abnormalities (hyperpigmentation, café-au-lait spots); deafness. Endocrinopathy affecting the pancreas (diabetes mellitus), growth hormone deficiency, and hypothyroidism; early menopause.
Hematological symptoms	Bone marrow failure or aplastic anemia typically starting at 5–10 years with thrombocytopenia. Exception: D1 and N patients may die before that age from AML or other childhood solid tumors (such as medullo- or nephroblastoma).
Cancer risk	800-fold increased risk of AML, mostly occurring at age 5–15 years, typically after the onset of marrow failure. At older ages there is a similarly increased risk of solid tumors, mainly carcinomas of the head and neck or oesophagus, as well as, in females, the vulva and vagina. D1 and N patients typically develop malignancies during early childhood (<5 years).
Overlapping syndromes**	Inherited bone marrow failure syndromes: Dyskeratosis congenita, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, severe congenital neutropenia, thrombocytopenia absent radii (TAR) syndrome, amegakaryocytic thrombocytopenia.Other overlapping syndromes: Baller-Gerold syndrome, Nijmegen breakage syndrome, Rothmund-Thomson syndrome, Roberts syndrome, Warsaw Breakage syndrome, DK-phocomelia, VACTERL hydrocephalus syndrome, Wiskott-Aldrich syndrome.

*Many symptoms show highly variable penetrance. In a sizable proportion of patients (ca. 30%), congenital abnormalities may be absent altogether. Features in bold are most consistently associated with the FA phenotype.

**For an overview of the overlapping inherited bone marrow failure syndromes, see [5, 25]. For the other overlapping syndromes, the reader is referred to the OMIM database. Three overlapping syndromes may score positive in a chromosomal breakage test (italic): Nijmegen breakage syndrome [7–9], Roberts syndrome, and Warsaw Breakage Syndrome [10].

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 [4-6].

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 [7-9], 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, 12]. 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 [14-18]. 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, 19–21]. 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.

2. Methods and Results

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.

3. Discussion

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.

Cell cycle analysis via flow cytometry has been used as an alternative way to diagnose FA in skin fibroblasts [29], amniocytes [30], and peripheral blood mononuclear cells [31-34]. This test is based on the fact that cells from FA patients are hypersensitive towards DNA cross-linking agents and tend to be delayed and arrested with a 4c DNA content in the late S/early G2 phase of the cell cycle [35-38]. With the exception of overt leukemia and complete lymphocyte mosaicism, the cell cycle test reliably differentiates between FA and non-FA blood samples, including non-FA patients with aplastic anemia, Nijmegen breakage syndrome, Roberts syndrome, Baller-Gerold syndrome, VACTERL, and other thrombo- and erythropenia syndromes, as these conditions lack elevated G2-phase cell fractions [39]. For details of the cell cycle assay, readers are referred to the published protocols [39, 40].

FANCD2 western blotting is another alternative procedure to diagnose FA [40]. With this method stimulated T lymphocytes are tested for the occurrence of the ubiquitinated isoform of FANCD2, which readily reveals FA in cases where this isoform is lacking (subtypes A, B, C, D2, E, F, G, I, L, and M). This is a convenient alternative for diagnosing >90% of all FA patients. A disadvantage is that the subtypes with a defect downstream of FANCD2 ubiquitination (D1, J, M, N, O, P and possibly new subtypes) are not diagnosed as FA. In addition, true FA cases with significant lymphocyte mosaicism may also be missed by FANCD2 western blotting.

Why would a relatively laborious breakage test still be relevant now that next-generation sequencing (NGS) is available to determine mutations in FA genes? Two types of result from NGS would require assessment of the cross-linker sensitive cellular phenotype. First, unclassified sequence variations may be identified, whose pathogenic status remains uncertain until functionally tested. Second, if all known FA genes were found to be unaffected by mutations, a putative new FA gene may be found mutated. Proof of identity as a new FA gene requires the demonstration of cellular hypersensitivity to cross-linking agents and some form of functional test where introduction of a wild-type allele should correct the phenotype.