Fanconi anemia is associated
with a defect in the BRCA2
Bing Xia1,7, Josephine C Dorsman2,7, Najim Ameziane2,
Yne de Vries2, Martin A Rooimans2, Qing Sheng1, Gerard Pals2,
Abdellatif Errami3, Eliane Gluckman4, Julian Llera5,
Weidong Wang6, David M Livingston1, Hans Joenje2&
Johan P de Winter2
The Fanconi anemia and BRCA networks are considered
interconnected, as BRCA2 gene defects have been discovered
in individuals with Fanconi anemia subtype D1. Here we show
that a defect in the BRCA2-interacting protein PALB2 is
associated with Fanconi anemia in an individual with a new
subtype. PALB2-deficient cells showed hypersensitivity to
cross-linking agents and lacked chromatin-bound BRCA2; these
defects were corrected upon ectopic expression of PALB2
or by spontaneous reversion.
Fanconi anemia is a recessive chromosomal instability syndrome with
both autosomal and X-linked inheritance. Clinical features are diverse
and include progressive bone marrow dysfunction and an extremely
elevated cancer risk. Cells derived from individuals with Fanconi
anemia are hypersensitive to growth inhibition and chromosomal
breakage induced by DNA cross-linking agents such as mitomycin C
(MMC) and diepoxybutane. Eleven distinct genes have been identified
that, when defective, can cause Fanconi anemia, and all Fanconi
anemia gene products are thought to function in a common network
of genomic maintenance1,2. At least eight Fanconi anemia proteins
form a nuclear ‘core complex’ that catalyzes the activation of the
Fanconi anemia D2 protein (FANCD2) by monoubiquitina-
tion. BRCA2 (also known as FANCD1) and BRIP1 (also known
as FANCJ) function downstream and/or independently of this
This study began with a search for a gene defect in an unclassified
individual with Fanconi anemia, EUFA1341, who showed normal
monoubiquitination of FANCD2 and in whom we did not detect
pathogenic sequence alterations in BRCA2 and FANCJ. As no further
Fanconi anemia subtypes are known to operate downstream of
FANCD2 activation, we suspected this individual of representing a
new Fanconi anemia complementation group, designated subtype N.
PALB2 (partner and localizer of BRCA2) is a recently discovered
protein that interacts with BRCA2 and is important in determining
the localization and stability of BRCA2 in the nucleus5. HeLa cells
in which PALB2 expression is reduced by short interfering RNA show
an increased sensitivity to MMC, a hallmark of Fanconi anemia5.
Therefore, we considered PALB2 a candidate for the protein defective
As an initial test for a possible PALB2 defect, we visualized
PALB2 by protein blotting using antibodies raised against a central
region of PALB2 (amino acids 601–880). In contrast to cells from
individuals with Fanconi anemia subtype D1 or subtype I and in
contrast to control cells, we did not detect full-length PALB2 in
EUFA1341 lymphoblasts and fibroblasts (Fig. 1a). The amount of
BRCA2 in EUFA1341 lymphoblasts was much lower than in
control cells (Fig. 1b), consistent with the previous observation that
PALB2 promotes BRCA2 stability in the nucleus5. The lack of
full-length PALB2 protein and the reduced amount of BRCA2
suggested the existence of sequence alterations in the gene encoding
PALB2. Notably, in a phenotypically reverted (MMC-resistant) sub-
line of EUFA1341 lymphoblasts (designated ‘EUFA1341(R)’), we
found a normal amount of BRCA2 without the reappearance of
full-length PALB2 (Fig. 1b).
Four lines of evidence showed that a loss of PALB2 function caused
Fanconi anemia in individual EUFA1341. First, although total BRCA2
was not reduced in EUFA1341 fibroblasts (Fig. 1c), the protein was
mislocalized, being grossly depleted from the nuclear pellet (P100)
fraction (Fig. 1d). Consequently, Rad51 focus formation induced by
MMC treatment was impaired in these fibroblasts (Fig. 1e). Second,
introduction of wild-type PALB2 into these cells normalized (i) the
association of BRCA2 with the chromatin/nuclear matrix fraction
(Fig. 1d), (ii) the ability to form Rad51 foci (Fig. 1e) and (iii) the
sensitivity to MMC (Fig. 1f). Third, we found pathogenic mutations
in the gene encoding PALB2 in EUFA1341. Sequencing of genomic
DNA as well as cDNA from EUFA1341 uncovered an apparently
homozygous or hemizygous nonsense mutation in exon 4, leading to
the amino acid change Y551X (primers are listed in Supplementary
Table 1 online). As we detected this mutation only in the mother of
this individual, EUFA1341 was probably compound heterozygous for
a deletion on the paternal allele in the region of the exon 4 mutation.
This was confirmed by multiplex ligation-dependent probe amplifica-
tion (MLPA) analysis6(Supplementary Fig. 1 online). Fourth, cDNA
sequencing and MLPA analysis of the reverted cells uncovered a
secondary sequence alteration that restored part of the PALB2 ORF
and could explain recovery of PALB2 activity (Supplementary Fig. 1).
The corrective alteration deleted the premature stop-containing
Received 16 August; accepted 27 November; published online 31 December 2006; doi:10.1038/ng1942
1Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.2Department of Clinical Genetics, VU Medical
Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.3MRC-Holland BV, Hudsonstraat 68, 1057 SN Amsterdam, The Netherlands.4Bone Marrow
Transplant Unit, Ho ˆpital Saint-Louis, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France.5Departamento de Pediatria, Hospital Italiano de Buenos Aires,
Buenos Aires, Argentina.6Laboratory of Genetics, National Institute on Aging, US National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, USA.
7These authors contributed equally to this work. Correspondence should be addressed to J.P.d.W. (email@example.com) or D.M.L. (David_Livingston@dfci.harvard.edu).
NATURE GENETICS VOLUME 39 [ NUMBER 2 [ FEBRUARY 2007159
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exon 4 from the mRNA, resulting in an in-frame fusion of exons 3
and 5 (Fig. 2a). Subsequent genomic sequencing showed a 5,962-bp
deletion between Alu repeats in introns 3 and 4 of the maternal allele
that was probably generated by spontaneous Alu-mediated recombi-
nation7. Although genetic reversion of somatic cells has been attrib-
uted to several distinct genetic mechanisms8, the above-noted results
indicate that Alu-mediated recombination in cis can be added to this
list. Given the role of PALB2 in efficient homologous recombination5,
it remains to be determined whether Alu-mediated recombina-
tion is enhanced in PALB2-deficient cells (for example, by promo-
ting single-strand annealing as a compensatory event for reduced
Further studies showed the existence of truncated PALB2 proteins
in primary and reverted EUFA1341 cells. Using immunoprecipitation
MMC concentration (nM)
5-d survival (%)
Figure 1 Phenotype of PALB2-deficient cells from an individual with
Fanconi anemia is corrected by ectopic expression of PALB2.
(a) Immunoblotting with an antibody against residues 601-880 of PALB2
demonstrated the absence of full-length PALB2 from whole-cell extracts of
lymphoblasts and fibroblasts of individual EUFA1341. (b) The amount of
BRCA2 is low in EUFA1341 lymphoblasts and normal in reverted
EUFA1341(R) lymphoblasts. (c) EUFA1341 fibroblasts stably transduced
with empty pOZC vector or pOZC-PALB2 were analyzed for PALB2, BRCA2
and Rad51 expression by immunoblotting whole-cell extract (WCE). (d) The
same cells as in c were fractionated into S100 (soluble, containing
cytoplasmic and nucleoplasmic proteins) and P100 (pellet, containing
chromatin, nuclear matrix and insoluble proteins). Unlike in WCE and S100,
amounts of BRCA2 and Rad51 were grossly reduced in P100 of EUFA1341
fibroblasts and were restored upon reintroduction of PALB2. (e) EUFA1341
fibroblasts had a reduced capability to form MMC-induced Rad51 foci,
which was restored by introduction of PALB2 or PALB2D4. (f) EUFA1341
fibroblasts showed a substantially increased sensitivity to MMC that was
corrected by introduction of PALB2 or PALB2D4 but not by introduction of
PALB2(Y551X) or empty (pOZC) vector. The data presented are the averages
of three independent experiments performed in duplicate. This research was
carried out with written informed consent and after approval by the
Institutional Review board of the VU Medical Center.
HSC93 (WT)HSC93 (WT)
pOZC-PALB2 pOZ vector
Protein blot: α1–200
Protein blot: α601–880
789 10 11 12
PB: αFLAG (M2)
Figure 2 PALB2 protein analysis in EUFA1341 and EUFA1341(R) lymphoblasts. (a) We detected a premature stop (1802T-A, Y551X) in the cDNA and
genomic DNA from EUFA1341 and found an in-frame deletion of exon 4 in cDNA from EUFA1341(R). This cDNA encodes a variant of PALB2 (PALB2D4)
in which residues 1–70 are fused to residues 562–1186. (b) Whole-cell extracts of HSC93 (wild-type), EUFA1341 and EUFA1341(R) lymphoblasts were
immunoprecipitated with antibodies to PALB2 residues 1–200 (lanes 1–3 and 7–9) or 601–880 (lanes 4–6 and 10–12) and analyzed by protein blotting
with antibodies against BRCA2 (lanes 1–12), PALB2 residues 1–200 (lanes 1–6) or 601–880 (lanes 7–12). Antibody a1–200 immunoprecipitated a protein
(’X’; lane 2) in EUFA1341 cells that was detected when the same antibody was used for protein blotting, but BRCA2 was not coimmunoprecipitated. Both
a1–200 and a601–880 immunoprecipitated a slightly larger protein (‘Y’; lanes 3, 6, 9 and 12) in EUFA1341(R). This protein reacted with both antibodies
in protein blots and coimmunoprecipitated BRCA2. In addition, a601–880 recognized protein ‘Z’ (lane 11) in EUFA1341 cells. (c) We loaded the same
immunoprecipitates as in lanes 2 and 12 of b along with recombinant PALB2(Y551X) or PALB2D4, generated in HEK293T cells. These recombinant
proteins migrated at the same position as proteins X and Y. The identity of protein Z is unknown. (d) HEK293T cells were transfected with the indicated
plasmids. Immunoprecipitation with anti-Flag M2 agarose beads showed that recombinant PALB2D4 efficiently binds BRCA2.
160 VOLUME 39 [ NUMBER 2 [ FEBRUARY 2007 NATURE GENETICS
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
and protein blotting, we detected truncated PALB2 lacking residues
encoded by exon 4 (protein ‘Y’) in EUFA1341(R) cells, whereas a
protein comprising only the first 550 residues of PALB2 (protein ‘X’)
was present in primary EUFA1341 cells (Fig. 2b,c). In contrast to the
truncated protein in the primary cell line, the truncated PALB2
protein from the reverted cells did interact with BRCA2 (Fig. 2b,d),
which provides an explanation of why BRCA2 levels were restored in
these cells. This protein also re-established MMC-induced Rad51
focus formation (Fig. 1e) and corrected the MMC-hypersensitive
phenotype of EUFA1341 cells (Fig. 1f). Our results show that a
shortened PALB2 protein with a large internal deletion is functional,
whereas the N-terminal 550 residues of PALB2 cannot function alone.
These data link mutations in PALB2 to the new Fanconi anemia
subtype Nand justify FANCNas an alias for this gene. Like many other
individuals with Fanconi anemia, individual EUFA1341 presented
with skin, thumb, heart and kidney abnormalities and growth retarda-
tion (Supplementary Methods online). In addition, this individual
developed anemia at a very early age and died at the age of 2 from an
aggressive, rare type of endothelial cancer, a kaposiform hemangioen-
dothelioma9,10. Similar to Fanconi anemia associated with biallelic
mutations in BRCA2, Fanconi anemia caused by mutations in PALB2
might represent an extreme variant of this disorder, with respect to the
severity of the clinical phenotype, time of anemia onset and cancer
susceptibility11. As PALB2 is critical for the function of BRCA2 in
DNA repair and tumor suppression5, it could, in principle, also be a
tumor suppressor protein. Several family members of individual
EUFA1341 indeed developed tumors, and some of these tumors fall
into the recently proposed BRCA2 tumor spectrum (Supplementary
Fig. 2 online)12,13. Further studies are necessary to assess the potential
role of PALB2 mutations in sporadic and familial (childhood) cancer
in non–Fanconi anemia populations.
Note: Supplementary information is available on the Nature Genetics website.
We thank the family of individual EUFA1341 for contributing to this study,
M. Makiya and C. de Leon Lucero for help in the collection of clinical data and
J. Steltenpool and A. Oostra for expert technical assistance. The members of the
Livingston laboratory are grateful to A. D’Andrea and his colleagues at the Dana
Farber Cancer Institute for a number of useful discussions and for Fanconi
anemia-related reagents. This study was financially supported by the Netherlands
Organization for Health Research and Development (J.P.d.W. and N.A.); by
grants, including a breast cancer SPORE, from the National Cancer Institute
(D.M.L.); by the Breast Cancer Program of US Army Medical Research and
Materiel Command (B.X.); by the Intramural Research Program of the National
Institute on Aging, US National Institutes of Health (W.W.) and by the Fanconi
Anemia Research Fund (H.J.)
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Genetics website
Published online at http://www.nature.com/naturegenetics
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