The DNA helicase BRIP1 is
defective in Fanconi anemia
complementation group J
Marieke Levitus1,7, Quinten Waisfisz1,6,7, Barbara C Godthelp2,7,
Yne de Vries1, Shobbir Hussain3, Wouter W Wiegant2,
Elhaam Elghalbzouri-Maghrani2, Ju ˆrgen Steltenpool1,
Martin A Rooimans1, Gerard Pals1, Fre ´ Arwert1,
Christopher G Mathew3, Ma$gorzata Z Zdzienicka2,4,
Kevin Hiom5, Johan P De Winter1& Hans Joenje1
The protein predicted to be defective in individuals with
Fanconi anemia complementation group J (FA-J), FANCJ, is a
missing component in the Fanconi anemia pathway of genome
maintenance. Here we identify pathogenic mutations in eight
individuals with FA-J in the gene encoding the DEAH-box DNA
helicase BRIP1, also called FANCJ. This finding is compelling
evidence that the Fanconi anemia pathway functions through
a direct physical interaction with DNA.
Fanconi anemia is a recessively inherited chromosomal instability dis-
order characterized by developmental abnormalities, retarded growth,
bone marrow failure and a high risk of cancer. Cells from individuals
with Fanconi anemia feature hypersensitivity to bifunctional alkylating
(cross-linking) agents such as mitomycin C1. Eleven Fanconi anemia
complementation groups are currently distinguished (FA-A, FA-B,
FA-C, FA-D1, FA-D2, FA-E, FA-F, FA-G, FA-I, FA-J and FA-L), nine of
which have been connected to distinct genes (FANCA, FANCB,
FANCC, BRCA2 (also called FANCD1), FANCD2, FANCE, FANCF,
FANCG and FANCL). The genes defective in complementation groups
FA-I and FA-J have not yet been identified2. The precise mechanism
by which the Fanconi anemia pathway stabilizes the genome is not
known, because the known Fanconi anemia–associated proteins lack
domains that could provide a clue about their collective function.
After numerous unsuccessful attempts to identify the gene mutated
in FA-J (FANCJ) using a complementation cloning strategy1,3, we
attempted positional cloning (Supplementary Methods online). We
studied eight individuals with FA-J, four of whom are from genetically
informative families and two of whom are from a multiplex consan-
guineous family (Supplementary Fig. 1 online). We used DNA from
one of these individuals in a genome-wide scan using polymorphic
markers positioned B5 Mb apart. Chromosome 17 had the largest
region of homozygosity (Supplementary Fig. 2 online), and we studied
this region in more detail using the additional informative families with
FA-J. We also tested this chromosome for complementation of the
Fanconi anemia defect by microcell-mediated chromosome transfer4.
Of six clones that had taken up chromosome 17 material, five showed
complementation of the sensitivity of FA-J fibroblasts to mitomycin C,
MMC concentration (ng/ml)
Figure 1 Mapping of the candidate region for FANCJ. (a) Microcell-mediated
chromosome transfer of chromosome 17 complements mitomycin C (MMC)
sensitivity in hTert-immortalized fibroblasts from individuals with FA-J.
Clonogenic survival of wild-type (VH10/hTert) and FA-J (EUFA30/hTert)
immortal fibroblasts and six clones of EUFA30/AG656 fibroblasts with an
additional chromosome 17. Data represent means and standard errors of
the means from at least three experiments. (b) Chromosome 17 showing the
regions excluded by linkage analysis in the four informative families with
FA-J (left blocks) and (non)complementing clones (middle blocks). The
remaining candidate regions are depicted in the right blocks, with their
sizes and the numbers of predicted genes they contain indicated.
Published online 21 August 2005; doi:10.1038/ng1625
1Department of Clinical Genetics and Human Genetics, VU University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands.
2Department of Toxicogenetics, Leiden University Medical Center, Wassenaarseweg 72, NL-2333 AL Leiden, The Netherlands.3Division of Genetics and Molecular
Medicine, King’s College London, Guy’s Hospital, London, UK.4Department of Molecular Cell Genetics, Nicolaus Copernicus University, Collegium Medicum of
L. Rydygier, Bydgoszcz, Poland.5MRC Laboratory of Molecular Biology, Hills Road, CB2 2QH, Cambridge, UK.6Present address: Department of Hematology,
VU University Medical Center, Amsterdam, The Netherlands.7These authors contributed equally to this work. Correspondence should be addressed to H.J.
934VOLUME 37 [ NUMBER 9 [ SEPTEMBER 2005 NATURE GENETICS
© 2005 Nature Publishing Group http://www.nature.com/naturegenetics
whereas one clone did not (Fig. 1a). We mapped the chromosome 17 Download full-text
fragment boundaries in these clones. These boundaries, together with
the information obtained from the genome-wide screen and the three
remaining genetically informative families, narrowed the FANCJ can-
didate region to two subregions, encompassing 11.2 Mbp (between
D17S921 and D17S1294) and 36.4 Mbp (between D17S1293 and
D17S2059), respectively (Fig. 1b).
Of the 786 predicted genes present in the defined regions, BRIP1,
encoding a DEAH-box helicase5, was a good candidate because chicken
DT40 cells lacking BRIP1 expression have a Fanconi anemia–like
phenotype6. We sequenced this gene in families with FA-J and
identified mutations in all affected individuals (Table 1), which
segregated with disease status in informative families. We found the
recurrent nonsense mutation R798X in exon 17, which predicts a
truncated protein in which the helicase motif VI and the BRCA1-
interacting region are deleted. We found this mutation in five alleles
from four individuals of diverse geographic origin, suggesting that it
might be a hot spot or an ancient mutation. All other mutations were
private. We found three splice site mutations: IVS3+5G-T, present
homozygously in individuals EUFA543 and 381 from a consangui-
neous family; IVS17+2insT, present in individual EUFA696; and
IVS11–498A-T, present in individual EUFA1333. We sequenced
cDNA from individual EUFA543 and found a deletion of exon 3,
which encodes the helicase motif I. We also sequenced cDNA from
individual EUFA696 and found three species of FANCJ mRNA, two of
which lacked either exon 17 or 18, both leading to a frameshift
resulting in partial deletion of helicase motif VI. Splice site prediction
programs showed that this mutation compromises the normal exon 17
splice site. In individual EUFA1333, a deep intronic nucleotide sub-
stitution gave rise to a strong donor splice site according to splice site
prediction programs, which in cDNA resulted in insertion of a cryptic
exon of 963 bp. The remaining alterations were missense mutations
and a frameshift mutation, 2255delAA, leading to a premature stop
codon, 12 amino acids downstream and inside motif V. The missense
mutations were in or close to a helicase motif (Supplementary Fig. 3
online). Except for missense mutation 790C-T (R264W) in the
family of individual EUFA543, none of the sequence alterations
encountered were found in 200 chromo-
somes from a European control population.
Because 790C-T (R264W) was found once
in the control population, we consider this a
Western blots using antibodies against
BRIP1 showed the absence of a full-length
BRIP1 protein band in nuclear extracts from
cell lines carrying truncating mutations,
whereas an attenuated band was observed in
the cell line from individual EUFA776 with
two missense mutations (Supplementary Fig.
4 online). We conclude that the gene BRIP1
(now also called FANCJ) underlies FA-J.
The process that is primarily defective in
Fanconi anemia cells has thus far been elusive,
as the domain structures of the known Fan-
coni anemia–associated proteins have not
yielded functional clues. The DEAH-box
DNA helicase FANCJ and the recently discov-
ered protein FANCM seem to be the first
have a direct interaction with DNA7. FANCJ
is a member of the family of RecQ DEAH
helicases, which are capable of unwinding non-Watson-Crick DNA
structures, such as Holliday junctions, formed during homologous
recombination or during the resolution of stalled replication forks.
Defects in three of the five known human RecQ homologs, BLM, WRN
and RECQL4, result in chromosomal instability syndromes that are
associated with cancer predisposition8. FANCJ is a RecQ-like helicase
that unwinds DNA in 5¢-3¢ direction, like XPD, and may therefore,
like the RecQ homologs, be associated with DNA repair9. Furthermore,
this protein may have a direct link with homologous recombination
through its interaction with BRCA1 (ref. 10).
Accession codes. GenBank: BRIP1 mRNA, NM_032043. GenBank Protein:
Note: Supplementary information is available on the Nature Genetics website.
We thank the families with Fanconi anemia for cooperating in this study; the
treating clinicians (S.E. Ball, R. Barr, J. Burn, L. Pitcher, C. Pellegrini, J.M. Hows
and J. Bodurtha) for referring their patients; R.A. Weinberg and B. Klein for
providing hTert plasmid; C. van Berkel and E.H. Laghmani for technical
assistance; and R. Kanaar for the RAD51C construct. This study was financially
supported by the FA Research Fund, the Dutch Cancer Society, the Netherlands
Organization for Health Research and Development and the Medical Research
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 3 May; accepted 26 July 2005
Published online at http://www.nature.com/naturegenetics/
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Table 1 FANCJ mutations in individuals with FA-J
IndividualOrigin ExonMutation Type
between III + IV
V + VI
between II + III
aThe affected amino acid residue is listed first, followed by fs (to indicate a frameshift), X (to indicate a stop) and then a number
indicating how many more residues occur before the stop.bIndividuals 381 and EUFA543 are from a consanguineous marriage.
cThese individuals were previously reported to have FA-J2.dThis mutation resulted in a deletion of either exon 17 or exon 18.
eThis individual is heterozygous with respect to polymorphic repeat markers surrounding the gene but homozygous with respect
to polymorphic SNPs in BRIP1; no material was available from the parents. The mutation could therefore be homozygous or
hemizygous.fThis individual is an adopted child of unknown ancestry.
The research was carried out after approval by the Institutional Review Board (VU University Medical Center, Amsterdam), and
written consent was obtained from the subjects involved.
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© 2005 Nature Publishing Group http://www.nature.com/naturegenetics