Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
The genetic and molecular basis of Fanconi anemia
Introduction
When the Swiss pediatrician Guido Fanconi first described a family with physical abnormalities and aplastic anemia [1], he could not have realized that the bone marrow failure syndrome he had discovered would reveal an important cellular defense mechanism against genetic instability. Many years after Fanconi's report it became evident that defects in several genes can cause Fanconi anemia (Table 1). The affected genes all encode proteins in a cellular pathway that is activated when DNA replication becomes blocked and that regulates mechanisms leading to recovery from this harmful situation. An effective way to block DNA replication is by covalently connecting the two complementary DNA strands with a DNA cross-linking agent. It is this type of agent to which FA cells are exceptionally sensitive. Since DNA cross-linking agents like cisplatin are often used to treat cancer patients, Fanconi's discovery had still broader implications than he could have imagined. In this review we will outline how the FA genes were identified and what role these proteins may play in the FA pathway.
Section snippets
DNA cross-linker sensitivity, an important hallmark for FA cells
Almost 40 years after Fanconi's report it was Traute Schroeder who noticed spontaneous chromosomal breakage during routine cytogenetic analysis of FA patients [2], which classified FA as a chromosomal instability syndrome. Ten years later, Masao Sasaki discovered that the chromosomal abnormalities in FA cells were drastically enhanced by the DNA cross-linking agent mitomycin C (MMC) [3]. This observation has provided the basis for the widely used diagnostic chromosomal breakage test, as first
Complementation group D, the first link between FA and DNA repair
Positional cloning (Fig. 3), which also helped to identify FANCA [21], was successfully utilized to discover the gene defective in complementation group D, by Markus Grompe and co-workers. In 1995, microcell-mediated chromosome transfer had already shown that the MMC-sensitive phenotype of the FA-D cell line PD20 was restored to wild type levels by introducing chromosome 3 in these cells [22]. Several of the complemented PD20 clones lacked the q-arm of chromosome 3, indicating that the
Multiple FA proteins act in a nuclear core complex
While the connection between the FA pathway and DNA repair became more obvious after the identification of FANCD1 and FANCD2, it was still a mystery what the nuclear FANCA, -C, -E, -F, -G complex was actually doing and how it was involved in the monoubiquitination of FANCD2. To get a more complete picture of the core complex Weidong Wang and co-workers purified a protein complex from HeLa nuclear extracts using an antibody against FANCA and analyzed its components by mass spectrometry (Fig. 4).
FANCJ, a helicase downstream or independent of FANCD2
In 2005 the link between FA and DNA repair became more firmly established, when in the same Nature Genetics issue that described the identification of FANCM also the BRCA1-interacting DEAH helicase BRIP1 was reported as the gene affected in FA-J patients (see commentary [57]). FANCJ was identified by genetic linkage analysis and microcell-mediated chromosome transfer [58], [59], [60]. Genome-wide linkage analysis with polymorphic repeat markers on a single consanguineous FA-J patient revealed a
PALB2/FANCN, another FA protein identified by protein association
The purification of the FA core complex clearly showed that novel players in a given pathway can be identified by immunoprecipitation strategies. This approach was also critical in the identification of PALB2 as a new component in the downstream part of the FA/BRCA pathway. David Livingston and co-workers investigated the proteins that bind to BRCA2 in a large-scale co-immunoprecipitation experiment [69]. They identified, in addition to RAD51, a novel BRCA2-interacting protein, which turned out
FANCI, the monoubiquitinated partner of FANCD2
The most recent addition to the FA/BRCA pathway is FANCI, which has been discovered in 2007 by different strategies [73], [74], [75]. Genome-wide linkage analysis on four FA-I families resulted in four candidate regions for FANCI. From these regions candidate genes were selected by bioinformatics and data mining. In candidate KIAA1794 pathogenic mutations were found in all patients classified as FA-I by complementation analysis, including the reference patent for this group [73]. At the same
A model for the FA/BRCA pathway
Although there may still be more new players in the FA/BRCA pathway that remain to be identified [78], the field now faces the challenge to define the functional relationships amongst the FA proteins and to precisely determine how the pathway acts to protect the genome. Part of the picture has become clear in the past decade (Fig. 5).
The majority of the FA proteins function in a nuclear core complex and defects in one of each individual subunit affect the stability of the entire complex. The
Conflict of interest statement
Nothing to declare.
Acknowledgements
The work of our group has been supported by the Dutch Cancer Society, the Netherlands Organization for Health Research and Development and the FA Research Fund Inc.
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