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Fanconi Anemia (FA) is a rare autosomal recessive cancersusceptibility disorder characterized by cellularhypersensitivity to mitomycin C (MMC). Six FA geneshave been cloned, but the gene(s) corresponding to FAsubtypes B and D1 remains unidentified. Here we showthat cell lines derived from FA-B and FA-D1 patientshave biallelic mutations in BRCA2 and express truncatedBRCA2 proteins. Functional complementation of FA-D1fibroblasts with wild-type BRCA2 cDNA restores MMCresistance. Our results link the six cloned FA genes withBRCA1 and BRCA2 in a common pathway. Germlinemutation of genes in this pathway may result in cancerrisks similar to those observed in families with BRCA1 orBRCA2 mutations.

Fanconi Anemia (FA) is a rare autosomal recessive cancersusceptibility syndrome characterized by congenitalabnormalities, progressive bone marrow failure, and cellularhypersensitivity to DNA crosslinking agents, such as MMCand cisplatin (1, 2). FA patients often develop acute myeloidleukemia (AML), but also develop squamous cell carcinomas,frequently of the head and neck or of the gynecologic system(3). Whether heterozygote carriers of FA gene mutations havean increased cancer risk remains unknown (4).

At least eight distinct complementation groups of FA(A,B,C,D1,D2,E,F,G) have been defined by somatic cellfusion studies (5–7), and six FA genes have been cloned(A,C,D2,E,F,G). The six known FA proteins interact in acommon pathway (8). Five of the FA proteins (A,C,E,F,G)assemble in a multisubunit nuclear complex. In response toDNA damage (8) or during S phase of the cell cycle (9), thiscomplex activates the monoubiquitination of the downstreamD2 protein, thereby targeting D2 to BRCA1-containingnuclear foci. Biallelic mutation of an upstream FA genedisrupts the monoubiquitination of FANCD2, resulting in lossof FANCD2 foci and hypersensitivity to MMC.

Recent studies suggest genetic interactions among thebreast cancer susceptibility genes, BRCA1 and BRCA2, andthe FA genes. First, disruption of BRCA1 results in loss ofDNA damage-inducible FANCD2 foci, suggesting thatBRCA1 may act as an “organizer” of FA foci (8).Accordingly, the BRCA1 protein has a Ring Finger E3ubiquitin ligase domain and may ubiquitinate FANCD2 invivo (10). Second, BRCA1(-/-) or BRCA2(-/-) tumor cells

exhibit MMC hypersensitivity and chromosome instability(11-13), similar to the defects observed in FA cells (fig. S1).Functional complementation of BRCA2(-/-) cells with murinewild-type Brca2 restores MMC resistance (14). Third,targeted inactivation of the murine Brca2 gene, disrupting thecarboxy terminus of the BRCA2 protein but sparing theamino terminus, results in viable mice with an FA-likephenotype (i.e., small size, skeletal defects, hypogonadism,cancer susceptibility, chromosome instability, and MMChypersensitivity) (15, 16).

To investigate the relationship between BRCA genes andFA, we sequenced BRCA1 and BRCA2 in cells derived fromFA-B and FA-D1 patients (Table 1 and Fig. 1A). While noBRCA1 mutations were detected, biallelic mutations inBRCA2 were observed. A homozygous mutation (IVS19-1 Gto A) was detected in a BRCA2 splice acceptor site in the FA-D1 reference line, HSC62, predicted to result in partial orcomplete loss of exon 20. In another FA-D1 line, EUFA423,two definitive BRCA2 mutations were identified, 7691insAT(exon 15) and 9900insA (exon 27). These mutant alleles werenot detected in a screen of 120 random genomic DNAsamples from the general population. Both mutations createframe-shifts and are predicted to encode C-terminal truncatedBRCA2 proteins. The 9900insA mutant allele has previouslybeen identified in a breast cancer kindred (Table 1) (17).

Paradoxically, the FA-B reference line, HSC230, alsocontained two abnormal BRCA2 alleles. One mutant allelecontained a known 3033delAAAC frameshift mutation inexon 11, and the second allele contained the polymorphicstop codon (ter3326) in exon 27 (18). This latter allele hasbeen detected in approximately 1% of normal controls in theUSA population and is not associated with a strong cancerrisk (18). That FA-D1 and FA-B cells had biallelic mutationsin the same gene (BRCA2) suggests the possibility of intra-allelic complementation or phenotypic reversion to wild-type(6). Two additional cell lines, from FA patients of unassignedsubtype, had biallelic mutations in BRCA2 (Table 1).

We next examined BRCA2 protein expression in the FA-D1 and FA-B cell lines (Fig. 1, B to D). An antibody to thecarboxy terminus of BRCA2 (Ab-2) recognized full lengthBRCA2 (380 kD) in normal control lymphoblasts, HeLacells, and HSC62(FA-D1) (Fig. 1B, lanes 1,2,5). EUFA423cells expressed a truncated BRCA2 protein (230 kD, lane 4),and no BRCA2 was detected in CAPAN1 (19) or HSC230(FA-B) cells with this antibody (lanes 3,6). Reprobing with a

Biallelic Inactivation of BRCA2 in Fanconi AnemiaNiall G. Howlett,1 Toshiyasu Taniguchi,1 Susan Olson,2 Barbara Cox,2 Quinten Waisfisz,3 Christine de Die-Smulders,4 NicolePersky,1 Markus Grompe,2 Hans Joenje,3 Gerard Pals,3 Hideyuki Ikeda,5 Edward A. Fox,1 Alan D. D’Andrea1*

1Department of Pediatric Oncology, Dana-Farber Cancer Institute and Department of Pediatrics, Children’s Hospital, HarvardMedical School, 44 Binney Street, Boston, MA 02115, USA. 2Department of Molecular and Medical Genetics and Departmentof Pediatrics, Oregon Health Sciences University, Portland, OR 97201, USA. 3Department of Clinical Genetics and HumanGenetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands. 4Departmentof Clinical Genetics, Academic Hospital Maastricht, Maastricht, The Netherlands. 5Department of Pathology, Sapporo MedicalUniversity School of Medicine, S-1, W-17, Chuo-ku, Sapporo 060-8557, Japan.

*To whom correspondence should be addressed. E-mail: alan_dandrea@dfci.harvard.edu

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different antibody (Ab-1, see epitope in Fig. 1A) revealedexpression of BRCA2 in EUFA423 and HSC230 (Fig. 1C,lanes 4,6), suggesting that these BRCA2 polypeptides aretruncated at the C-terminus (BRCA2C’∆, 370 kD). Ab-1 alsorecognized the truncated BRCA2 protein (BRCA2-Trunc) inEUFA423 (lane 4), suggesting that this isoform has aninternal deletion between the two antibody epitopes. Takentogether, these results indicate that EUFA423 and HSC230express BRCA2 polypeptides (BRCA2C’∆) with small C-terminal truncations, consistent with the presence ofmutations in exon 27 (Table 1). Cell lines from other FAsubtypes displayed approximately equal levels of full lengthBRCA2 (Fig. 1D).

Although HSC62 cells express BRCA2 protein ofapproximately normal size, the mutation (IVS19-1 G to A)predicts the presence of an abnormally-spliced mRNA. Totest this, we performed RT-PCR of the BRCA2 mRNA,followed by direct cDNA sequencing (Fig. 2, A to C). As aresult of this mutation, the BRCA2 mRNA lacks the first 12bases of exon 20, corresponding to an in-frame deletion offour amino acids from BRCA2 (a.a. 2830 to 2833) (Fig. 2C)(fig. S2). No normal BRCA2 mRNA was detected in HSC62cells. The mutant protein may have partial activity, since theHSC62 patient has a relatively mild clinical FA phenotype(table S1) and the HSC62 cells have only modest MMCsensitivity (20) (Table 2).

We next determined whether the BRCA2 mutant allelessegregate in the EUFA423 kindred (Fig. 3), by means ofgenomic PCR with specific flanking primers and directsequencing. The paternal allele was 7691insAT and thematernal allele was 9900insA (Fig. 3A). The proband(EUFA423) was a compound heterozygote, while two of thethree unaffected siblings were BRCA2 carriers. Lymphoblastsfrom all heterozygous BRCA2 carriers expressed full-lengthBRCA2 (Fig. 3B).

We next stably transfected EUFA423 fibroblasts withcDNA encoding the full-length wild-type BRCA2 protein(Fig. 3C). G418-selected cells expressed full-length BRCA2(Fig. 3C, lane 3) and exhibited a correction of their MMCsensitivity (Table 2). Similarly, transfection with humanchromosome 13, containing the wild-type BRCA2 gene,corrected the MMC hypersensitivity (Table 2). Takentogether, these results confirm that BRCA2 is an FA gene.

FA has an estimated incidence of less than 1 per 100,000live births, and less than 5% of FA families are assigned tosubtypes B and D1. BRCA2 mutations have a cumulativecarrier frequency of approximately 1% of the U.S. population(17). This BRCA2 carrier frequency predicts a higherincidence of BRCA2 homozygotes than the observed FAincidence. Based on our limited sample collection, FApatients have at least one mutation in the 3’ region of BRCA2.Thus, only a subset of BRCA2 (-/-) individuals (namely, thoseexpressing truncated BRCA2 proteins with partial activity)may manifest the FA phenotype. Homozygous disruption ofthe 5’ end of BRCA2, in contrast, may result in embryoniclethality, similar to the outcome in the mouse model (21-23).

Specific BRCA2 mutations may vary in cancer risk (17).The 6174delT mutation found in Ashkanazi-Jews may confera breast cancer risk as high as 70% by age 70. Other variantBRCA2 alleles, such as the polymorphic stop codon ter3326,appear to have no increased cancer risk (18), but may causeFA in the compound heterozygous state. The smallest knowncancer-associated deletion removes only 224 amino acidsfrom the C-terminus of BRCA2 (24). Due to theunavailability of clinical records, we were unable to assess

the cancer risk of the BRCA2 mutant alleles in these FAfamilies (Table 1).

FA patients with biallelic BRCA2 mutations share clinicalfeatures with FA patients from other subtypes (i.e., congenitalabnormalities, abnormal skin pigmentation, bone marrowfailure, and cellular sensitivity to MMC) (25) (table S1).These similarities suggest that BRCA2 and other FA proteinscooperate in a common DNA damage response pathway, theFA/BRCA pathway (Model, fig. S3A). According to thismodel, DNA damage activates the monoubiquitination ofFANCD2, thus targeting FANCD2 to DNA repair focicontaining BRCA1 and BRCA2 (26). Previous studies haveindicated that FA-B cells lack FANCD2 monoubiquitinationwhile FA-D1 cells express monoubiquitinated FANCD2 (8)(fig. S3B). BRCA2 may function upstream in the pathway, bypromoting FA complex assembly and FANCD2 activation,and/or downstream in the pathway, by transducing signalsfrom FA proteins to RAD51 and the homologousrecombination machinery (27). The precise molecularfunction(s)of BRCA1 and BRCA2 in this pathway remain tobe elucidated.

References and Notes1. M. Grompe, A. D'Andrea, Hum. Mol. Genet. 10, 2253.

(2001).2. H. Joenje, K. J. Patel, Nature Rev. Genet. 2, 446 (2001).3. B. P. Alter, Am. J. Hematol. 53, 99 (1996).4. M. Swift, R. J. Caldwell, C. Chase, J. Natl. Cancer Inst.

65, 863 (1980).5. H. Joenje et al., Am. J. Hum. Genet. 61, 940 (1997).6. H. Joenje et al., Am. J. Hum. Genet. 67, 759 (2000).7. C. Timmers et al., Mol. Cell 7, 241 (2001).8. I. Garcia-Higuera et al., Mol. Cell 7, 249 (2001).9. T. Taniguchi et al., Blood In Press (2002).10. C. A. P. Joazeiro, A. M. Weissman, Cell 102, 549 (2000).11. K. J. Patel et al., Mol. Cell 1, 347 (1998).12. A. Tutt et al., Curr. Biol. 9, 1107 (1999).13. M. E. Moynahan, T. Y. Cui, M. Jasin, Cancer Res. 61,

4842 (2001).14. M. Kraakman-van der Zwet et al., Mol. Cell. Biol. 22, 669

(2002).15. F. Connor et al., Nature Genet. 17, 423 (1997).16. K. A. McAllister et al., Cancer Res. 62, 990 (2002).17. J. Natl. Cancer Inst. 91, 1310 (1999).18. S. Mazoyer et al., Nature Genet. 14, 253 (1996).19. M. Goggins et al., Cancer Res. 56, 5360 (1996).20. C. A. Strathdee, A. M. V. Duncan, M. Buchwald, Nature

Genet. 1, 196 (1992).21. A. Suzuki et al., Genes Dev. 11, 1242 (1997).22. T. Ludwig, D. L. Chapman, V. E. Papaioannou, A.

Efstratiadis, Genes Dev. 11, 1226 (1997).23. S. K. Sharan et al., Nature 386, 804 (1997).24. S. Hakansson et al., Am. J. Hum. Genet. 60, 1068 (1997).25. M. Buchwald, J. Ng, C. Clarke, G. Duckworth-Rysiecki,

Mutat. Res. 184, 153 (1987).26. T. Taniguchi et al., Cell 109, 459 (2002).27. A. A. Davies et al., Mol. Cell 7, 273 (2001).28. J. Chen et al., Mol. Cell 2, 317 (1998).29. F. Xia et al., Proc. Natl. Acad. Sci. U S A 98, 8644

(2001).30. We thank J.Garber, F. Li, D. Livingston, D. Silver, S.

Meyn, D. Pellman, and M. Buchwald for helpfuldiscussions, J. Chen for the BRCA2 cDNA, and L. Moreaufor chromosome breakage analysis. Supported by NIH

/ www.sciencexpress.org / 13 June 2002 / Page 3/ 10.1126/science.1073834

grants RO1HL52725, RO1DK43889, and PO1HL54785(A.D.D.) and the Fanconi Anemia Research Fund.

Supporting Online Material(www.sciencemag.org/cgi/content/full/1073834/DC1)Materials and Methodsfigs. S1 to S3table S1

10 May 2002; accepted 3 June 2002

Published online 13 June 2002;<zdoi;10.1126/science.1073834>

Include this information when citing this paper.

Fig. 1. FA-B and FA-D1 cells have biallelic BRCA2mutations and express mutant BRCA2 proteins. (A)Schematic diagram of human BRCA2. The highly conservedBRC repeats, encoded by exon 11, mediate RAD51interactions. BRCA2 mutations in EUFA423 (FA-D1),HSC62 (FA-D1), and HSC230 (FA-B) lymphoblasts areshown. Mutations were confirmed in primary cells. (B)Whole cell lysates were prepared from wild-type PD7lymphoblasts, HeLa cells, CAPAN1, EUFA423 (FA-D1),HSC62 (FA-D1), and HSC230 (FA-B) lymphoblasts.CAPAN1 is a pancreatic carcinoma cell line which has lostone BRCA2 allele and contains the 6174delT mutation in theremaining allele (19). Proteins were separated byelectrophoresis and immunoblotted with a rabbit polyclonalantibody anti-BRCA2 (raised against amino acids 3245-3418of BRCA2) (Ab-2, Oncogene Research) or (C) a mousemonoclonal anti-BRCA2, raised against amino acids 1651-1821 (Ab-1, Oncogene Research). A protein in HeLa cellextracts (209 kD) was nonspecific. (D) Characterization ofthe BRCA2 protein in FA lymphoblasts from multiplecomplementation groups (subtypes A, C, D1, D2, E, F andG). Proteins were immunoblotted with Ab-2. (S, MMCsensitive; R, MMC resistant)

Fig. 2. The FA-D1 reference line, HSC62, expresses aBRCA2 protein with an internal deletion of four amino acids.(A) Schematic representation of the RT-PCR reaction,resulting in specific amplification of a region of the BRCA2mRNA. PCR products from the indicated cell lines wereanalyzed on a 1% agarose gel. (B) PCR products wereanalyzed by direct DNA sequencing. (C) BRCA2 mRNAfrom HSC62 cells has an internal deletion of the first twelvebases from exon 20, resulting in an in-frame deletion of theindicated four amino acids.

Fig. 3. Segregation of BRCA2 mutant alleles in the EUFA423pedigree. (A) The proband with FA subtype D1 is EUFA423.Genomic DNA was prepared from lymphoblasts from theindicated family members and sequenced for BRCA2mutations. (B) Expression of mutant BRCA2 polypeptides inlymphoblasts derived from EUFA423 kindred. Proteins wereimmunoblotted with Ab-2. (C) EUFA423F was transfectedeither with pcDNA3-empty vector or pcDNA3-HA-BRCA2(28) (29), and stable G418-resistant cells were isolated. Celllines were analyzed by immunoblot with Ab-2 and by theMMC chromosome breakage assay (Table 2).

Tab

le 1

. FA

pat

ient

s w

ith B

iall

elic

Mut

atio

ns in

BR

CA

2

Cel

l lin

eF

A S

ubty

peA

ssig

nmen

tM

utan

tA

llele

#1

(exo

n)

BIC

entr

yM

utan

tA

llele

#2

(exo

n)

BIC

entr

y

HS

C62

D1

IVS1

9-1

G to

A(2

0)

-IV

S19-

1 G

to A

†(2

0)

-

EU

FA42

3D

176

91 in

sAT

(15)

-99

00 in

sA(2

7)4

HSC

230

B30

33 d

elA

AA

C(1

1)

man

y10

204

A to

T‡

(27)

man

y

EU

FA

579

U/A

*72

35 G

to A

(13)

158

37T

C to

AG

(11)

1

AP

37P

U/A

*84

15 G

to T

(18)

287

32 C

to A

(20)

1

* U

/A, u

nass

igne

d FA

sub

type

† F

amily

His

tory

of

Con

sang

uini

ty

‡ P

olym

orph

ic S

TO

P va

rian

t (te

r332

6)

BIC

, Bre

ast C

ance

r In

form

atio

n C

ore

(ww

w.n

hgri

.nih

.gov

/intr

amur

al_r

esea

rch/

lab_

tran

sfer

/bic

)

Tab

le 2

. C

hrom

osom

e br

eaka

ge a

naly

sis

of F

A a

nd c

ontr

ol c

ell l

ines

. G

roup

s of

exp

erim

ents

are

sep

arat

ed b

y lin

e sp

aces

. S, M

MC

-se

nsiti

ve; R

, MM

C-r

esis

tant

; L, E

BV

-tra

nsfo

rmed

lym

phob

last

s; F

, SV

-40

tran

sfor

med

fib

robl

asts

. N

D, n

ot d

eter

min

ed.

Chr

omos

ome

brea

kage

ana

lysi

s w

as p

erfo

rmed

as

in (

7).

Cel

l lin

e/H

ybri

ds

MM

C (

ng/m

l)

%C

ells

wit

h R

adia

ls

Phen

otyp

e

Lym

phob

last

sPD

7L (

wt)

208

RE

UFA

121L

(F

A-F

)20

45S

HS

C62

L (

FA

-D1)

2038

SE

UFA

423L

(F

A-D

1)20

91S

HSC

230L

(F

A-B

)20

94S

PD7L

(w

t)20

2R

EU

FA12

1L (

FA

-F)

2064

SE

UFA

423L

(F

A-D

1)20

90S

EU

FA42

4L20

8R

EU

FA42

5L20

0R

EU

FA66

4L20

4R

EU

FA66

5L20

6R

EU

FA66

6L20

ND

Fib

robl

asts

E

xper

imen

t I

II

GM

0637

F (

wt)

2510

6

R

GM

6914

F (

FA

-A)

2564

49

S

EU

FA42

3F (

FA

-D1)

+ p

cDN

A3-

empt

y25

8891

SE

UF

A42

3F (

FA

-D1)

+ p

cDN

A3-

HA

-BR

CA

225

2428

RE

UFA

423F

+ H

uman

Chr

omos

ome

13

259

15R