Research Article: Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

Date Published: June 21, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Johan J. P. Gille, Karijn Floor, Lianne Kerkhoven, Najim Ameziane, Hans Joenje, Johan P. de Winter.


Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.

Partial Text

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: FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, and -P. [1–4]. The majority of patients (~85%) belong to the subtypes A (~60%), C (~10–15%), or G (~10%), while a minority (~15%) is distributed over the remaining 12 subtypes, with relative prevalences between <1 and 5%. These percentages may differ considerably within certain ethnic groups, due to founder effects. All subtypes seem to fit within a “classical” FA phenotype, except for D1 and N (mutated in BRCA2/FANCD1 and PALB2/FANCN), which are associated with a distinct, more severe, syndromic association. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. These two different modes of inheritance have important consequences for the counseling of FA families. The relative prevalence of FA-B amongst unselected FA patients is estimated at 1.6% [5]. For all genetic subtypes disease genes have been identified (Table 1). Many mutations found in the various subtypes are private, but recurrent mutations are known, particularly in specific ethnic backgrounds (Table 2). Cells derived from FA patients are—by definition—hypersensitive to chromosomal breakage induced by DNA cross-linking agents such as mitomycin C (MMC) or diepoxybutane (DEB) [31]. This cellular phenotype is ascertained using stimulated blood T lymphocytes. The indications for FA laboratory testing are rather broad [32]. As a consequence, in only a small proportion of patients (about 10%) the chromosomal breakage test is positive, and an FA diagnosis is established. Since mutation testing by Sanger sequencing and MLPA is rather laborious, time consuming and therefore expensive, a positive chromosomal breakage test is a prerequisite for starting mutation screening. Confirmation of the FA diagnosis at the DNA level is important in patients in whom the chromosomal breakage test was inconclusive. Furthermore, knowledge about the FA subtype is relevant for the treatment and prognosis of the patients. In addition, identification of mutations allows carrier testing in the family and will enable prenatal DNA testing and preimplantation genetic diagnosis (PGD) in future pregnancies. Finally, this information can be used to rule out FA in potential donors for bone marrow transplantation.   Source:


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