Supplementary Material
PCR amplification of the BRCA2 gene
The components of the PCR reaction were: 20mM Tris-HCl(pH8.4), 50mM KCl, 1.5mM
MgCl2, 0.1mM in each of dATP, dCTP, dGTP, TTP, 0.1µM of each primer, 5ng/µl DNA,
0.05units/µl Taq polymerase (Taq Platinum, GIBCO BRL, Gaithersburg, MD). The
primer sequences are as follows:
Exon
Primer name
2
2-F
2-R
3-F
3-R
4-F
4-R
5/6-F
5/6-R
7-F
7-R
8-F
8-R
9-F
9-R
10a-F
10a-R
10b-F
10b-R
10c-F
10c-R
10d-F
10d-R
10e-F
10e-R
11a-F
11a-R
11b-F
11b-R
11c-F
11c-R
11d-F
11d-R
11e-F
3
4
5/6
7
8
9
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
Sequence
GTT CCA GGA GAT GGG ACT GAA
ACA CAT AAG GAA CAG TTT ATG GTT
CCA TAG TCA AGA TCT TTA GCA
ACT GAT TTG CCC AGC ATG ACA
TTA CAA CTC CCT ATA CAT TCT CA
AAC CAG CCA ATT CAA CAT CAC A
ATA TCT AAA AGT AGT ATT CCA ACA A
AAT TGC CTG TAT GAG GCA GAA T
GTT ATA CCT TTG CCC TGA GAT T
GTC AGT TAC TAA CAC ACT TAT CA
GTT TAT TCA CTG TGT TGA TTG AC
CAT ATA GGA CCA GGT TTA GAG A
CAT CAC ACT ACT CAG GAT GAC A
GCA TGG TGG TGC ATG CTT GTA
CCA AGT ACT CAG AAT AAC CCT T
TTT GTC ACT TCC ACT CTC AAA G
TCC ATG AAG CAA ACG CTG ATG A
CCA GAT ATT GCC TGC TTT ACT G
GAC CTA TTA GAC ACA GAG AAC A
CTG CAT TCT TCA AAG CTA CAG A
TCA GGT CAT ATG ACT GAT CCA A
AAC ACA GAA GGA ATC GTC ATC T
CCG AAA GAC CAA AAA TCA GAA CT
AGC AAA CCA ACA TGG CAT ACG T
CCA AAC ACT ACC TTT TTA ACT TAG T
GAC CTC TTC TTT TAT ATC TGA AAC T
CTG AAG AAC CAA CTT TGT CCT TA
AGT GCT GGC ATT TTC ATG ATC AT
TAT TAC CCC AGA AGC TGA TTC T
TAC CTT TGA GCT TGT CTG ACA T
ATG TCA CCC AGT ACA ACA TTC A
CCT TTC ATT AGC AAC TTG GAA GA
GAG AGT AGC ATC ACC TTC AAG A
Amplicon size
348 bp
466 bp
454 bp
421 bp
398 bp
372 bp
495 bp
497 bp
470 bp
454 bp
416 bp
400 bp
380 bp
350 bp
299 bp
484 bp
464 bp
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
11w
12
13
14a
14b
15
16
11e-R
11f-F
11f-R
11g-F
11g-R
11h-F
11h-R
11i-F
11i-R
11j-F
11j-R
11k-F
11k-R
11l-F
11l-R
11m-F
11m-R
11n-F
11n-R
11o-F
11o-R
11p-F
11p-R
11q-F
11q-R
11r-F
11r-R
11s-F
11s-R
11t-F
11t-R
11u-F
11u-R
11v-F
11v-R
11w-F
11w-R
12-F
12-R
13-F
13-R
14a-F
14a-R
14b-F
14b-R
15-F
15-R
16-F
16-R
CTG CCC ATT TGT TCA TGT AAT C
AAA CAA GCA ACC CAA GTG TCA A
CAG AAA CAA CTA CAC TAC TCT GT
GGA AAT CAA GCT CTC TGA ACA T
CAT CTG GTT TTC AGG CAC TTC A
CCC CTC AGA TGT TAT TTT CCA A
ACC CCA CTT CAT TTT CAT CTG T
GGA ATG CAG AGA TGC TGA TCT
CAT TGA AAC GAC AGA ATC ATG AC
CTG CTC ATG GCA CAA AAC TGA
GAA TTT CTA CTG GCA GCA GTA T
CTT CTG CAG AGG TAC ATC CAA
TGC TCC GTT TTA GTA GCA GTT A
CTG ATC AGC ACA ACA TAT GTC T
CTT TTC ATC ACG TTC GGG TTG T
GAT CAG AAA CCA GAA GAA TTG C
ACA CTT TGG GGC AGC TGT GAT
TGC AAA GGA ATC TTT GGA CAA AG
GCT AAG GCT GAA TTT TCA ATG AC
GTG GTG CCA CCT AAG CTC TTA
GTA TCT TGT TTT TCG GAG AGA TG
ACT TCT GTG AGT CAG ACT TCA T
TGT GGG TAT GCA TTT GCA TCT T
CAG TAG CAT GTC TAA CAG CTA T
GTT TCA TGT GAA ACA CAA ACG AT
GAT ATT TGC GTT GAG GAA CTT G
GCG TGC TAC ATT CAT CAT TAT C
GGA TAG CCA GTG GTA AAA TCG T
AGA CTT GCT TGG TAC TAT CTT CT
TCA CCT TGT GAT GTT AGT TTG GA
CAT TTT GTC TAG ACG TAG GTG AA
CTG CTA TAC GTA CTC CAG AAC A
AAC AAG TGA GAC TTT GGT TCC TA
GCA CTG TGT AAA CTC AGA AAT G
TGT GGC ATG ACT TGG CAG TTT
ACT TTT TCT GAT GTT CCT GTG AA
TCC CCC AAA CTG ACT ACA CAA
CAT TTA AAG AGT CAA TAC TTT AGC T
GCA CAG TGG CTC ATG TCT GTA
GCA TCC GTT ACA TTC ACT GAA A
TAA CTT CTT AAC GTT AGT GTC ATT
AGG CTA GCC TTG AAA AAT GTG A
CAT CAG AGC GAT GTC CAT CAA
TTG ATT ACT ACA GGC AGA CCA A
TAA CAA GTC CAC AAG AAG TTA TC
TTT TGG TCA GGC TGG TCT TGA
TCA TTC ATC CAT TCC TGC ACT AA
TTT TGT AGT GAA GAT TCT AGT AGT
CAG AAT GCT TAA CCA TAA TGC AC
433 bp
403 bp
387 bp
346 bp
331 bp
496 bp
470 bp
423 bp
408 bp
386 bp
371 bp
348 bp
328 bp
498 bp
469 bp
431 bp
406 bp
383 bp
332 bp
302 bp
495 bp
449 bp
482 bp
472 bp
17
18a
18b
18c
19
20
21
22
23
24
25a
25b
26
27a
27b
27c
27d
17-F
17-R
18a-F
18a-R
18b-F
18b-R
18c-F
18c-R
19-F
19-R
20-F
20-R
21-F
21-R
22-F
22-R
23-F
23-R
24-F
24-R
25a-F
25a-R
25b-F
25b-R
26-F
26-R
27a-F
27a-R
27b-F
27b-R
27c-F
27c-R
27d-F
27d-R
CAC CAT GCT CAG CAA TGA AGT
GAT GGC AAC TGT CAC TGA CAA
TCC ACT ATT TGG GGA TTG CTA A
TAC CAC CCA TCT GTA AGT TCA A
TAG AAG CAG AAG ATC GGC TAT A
GAA TTT AAC TGA ATC AAT GAC TGA
TCC TCC CCT CTT AGC TGT CTT
GAC CTC CCA AAA ACT GCA CAA A
GGC AGT TCT AGA AGA ATG AAA AC
ACC CCT TCT CTA CCA AAA ATA CA
CTC AGG TGA TCC ACT AAT CTC
CCC TTG TTG CTA TTC TTT GTC T
TGA CAG AGT GAG ACC CTG TCT
CCT TTT GGA GAA ATG CAG CAT T
CAC ACC CTT AAG ATG AGC TCT
TAG TGG ATT TTG CTT CTC TGA TA
ATC CAC TAC TAA TGC CCA CAA A
TCC CGT GGC TGG TAA ATC TGA
ACA TAC AGT TAG CAG CGA CAA A
CAG ATC ACT AGT TAG CTA GCA A
AGC TTT CGC CAA ATT CAG CTA T
CTC TTG AAA GTG GCC CTC TTT
GGA TAG ACC TTA ATG AGG ACA T
TCC TGA GGT TCA TGG GCA ATT
GCA TGT TTG ACA ATT GGT ATC AC
GGA GCC ACA TAA CAA CCA CAT T
GGG GAG GGA GAC TGT GTG TA
TTT CGT ATT TGG TGC CAC AAC T
AAG TCT TGT AAA GGG GAG AAA GA
CTG GTG GGA GCA GTC CTA GT
ATT CTC CTC AGA TGA CTC CAT T
ACT GGA AAG GTT AAG CGT CAA T
CTC AGA CTG AAA CGA CGT TGT A
GCA ACT GAA GCA AAA GTA TAC CA
498 bp
432 bp
389 bp
312 bp
480 bp
453 bp
411 bp
443 bp
416 bp
398 bp
361 bp
336 bp
427 bp
400 bp
379 bp
352 bp
318 bp
One primer (designated “-F”) in each pair was synthesized with an 18base M13
–21 forward sequence (TGTAAAACGACGGCCAGT) at its 5’ end, and the other primer
(designated “-R”) was synthesized with an 18 base M13 –28 reverse sequence
(CAGGAAACAGCTATGACC) at its 5’ end. For the longer exons, two or more
overlapping amplicons were designed.
The thermocycling conditions were: 94˚C, 4min, followed by 11 cycles, each
with a denaturing step at 94˚C for 20 seconds and an extension step at 72˚C for 20
seconds, and with a 20 second annealing step that decreased 1˚C/ cycle, beginning at
60˚C in the first cycle and decreasing to 50˚C in the eleventh cycle; the eleventh cycle
was then repeated 25 times; a 6 minute incubation at 72˚C followed by a 4˚C soak
completed the program.
DNA sequencing
An aliquot of each PCR reaction was diluted 1:10 with water. The diluted PCR product
was sequenced on both strands using an M13 Forward and an M13 Reverse Big Dye
Primer kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s
recommendations. The sequencing products were separated on a fluorescent sequencer
(model 377 from Applied Biosystems, Foster City, CA). Base calls were made by the
instrument software, and reviewed by visual inspection. Each sequence was compared to
the corresponding normal sequence using Sequencher 3.0 software (LifeCodes, Stamford,
CT).
Denaturing Gradient Gel Electrophoresis (DGGE) analysis of exon 15 and 27 of the
BRCA2 gene
Genomic DNA samples were from healthy blood donors derived from the Netherlands
Center of Blood Transfusion Service (CLB, Amsterdam, The Netherlands). DNA
fragments that harbor the mutations in the EUFA423 patient, exon 15 and exon 27, were
amplified from the patient and 60 unrelated controls. Primer sets, IC-B2-15.1F with ICB2-15.1R, and IC-B2-27.1F with IC-B2-27.1R, were obtained from Ingeny (Ingeny
International, The Netherlands) (www.Ingeny.com). Samples were run in parallel using
DGGE (1). Gels were stained with ethidium bromide and visualized with UV light.
Reverse Transcription-PCR
For RT-PCR analysis of the HSC62 cells, RNA was purified from fibroblasts and
lymphoblasts using the Qiagen RNeasy Protect mini kit. First-strand cDNA was
synthesized from 2 µg RNA using Superscript II reverse transcriptase (Invitrogen,
Carlsbad, CA). Primers used to amplify a region of BRCA2 from exon 18 through exon
22 were BRCA2ex18FP (CCTCCCCTCTTAGCTGTCTTAAA) and BRCA2ex22RP
(CCCTTGATAAACCTTGTTCCTTT). Primers used to amplify a region of the
FANCD2 gene were DF3862 (CATCCTGTTCTGCATGTATG) and DSR4360
(TGATGACTCTGATTAGACCC).
Segregation analysis of EUFA423 kindred
For segregation analysis of EUFA423 pedigree, DNA from lymphoblastoid cell lines of
the father (EUFA424L), mother (EUFA425L) and three siblings (EUFA664L,
EUFA665L and EUFA666L) of EUFA423 were used to PCR amplify exon 15 and region
27a in exon 27 of the BRCA2 gene. The DNA sequence of both strands of each PCR
product was determined.
Microcell mediated chromosome transfer of human chromosome 13 into EUFA423
fibroblasts.
Microcell fusions were performed as described (2). Briefly, donor A9+13 Hytk cells
(mouse A9 cells containing hygromycin-marked human chromosome 13 (3) were split
into 150 mm2 tissue culture plates. After 20-24 h, micronuclei were induced by treating
the A9+13 Hytk cells for 48 h with 0.05 mg/ml colcemid. Micronucleated cells were
then trypsinized and allowed to sit for 8-16 h onto tissue culture plates cut into the shape
of a bullet. Bullets were then placed into 50 ml centrifuge tubes containing enucleation
media (serum-free alpha-MEM and 10 mg/ml of cytochalasin B) and centrifuged at
14,000 rpm for 30 min at 37˚C. The resulting microcell pellets were resuspended in
serum-free alpha-MEM and filtered through an 8 m and then a 5 m Whatman Nuclepore
membrane. The filtered microcells were then mixed with 100 mg/ml of
phytohemagglutinin P and added to a monolayer of immortalized EUFA423 fibroblasts.
After 15 min fibroblasts and A9+13 Hytk microcells were fused with 50% polyethylene
glycol for 1 min, washed with serum-free alpha-MEM and allowed to grow overnight in
alpha-MEM with 15% fetal bovine serum. The next day, cells were split 1:5 onto 150
mm2 tissue culture plates, and the following day, cells were selected in alpha-MEM
medium, supplemented with 15% fetal bovine serum, 200 µg/ml hygromycin and
hypoxanthine, aminopterin, thymidine (HAT). Clones were subsequently picked,
expanded and analyzed.
Chromosome Breakage Analysis
Chromosome Breakage Analysis was performed as previously described (4).
fig. S1. FA-D1 cells and BRCA2(-/-) tumor cells exhibit a similar pattern of
chromosome breakage. Metaphase chromosome spreads from FA-D1 fibroblasts or
CAPAN1 cells exposed to MMC (20 ng/ml) for 48 hours. Radial forms are indicated
(arrows).
fig. S2. The FA-D1 reference line, HSC62, contains a homozygous mutation in the
splice acceptor site of intron 19 (IVS19-1 G to A). Schematic representation of an
alternate splicing mechanism at the junction of intron 19 and exon 20 of HSC62,
resulting in the loss of the first 12 bases of exon 20, corresponding to an in-frame
deletion of four amino acids from BRCA2 (a.a. 2830 to 2833).
fig. S3. Possible functions of the BRCA2 protein in the FA/BRCA pathway. (A)
Schematic representation of the FA/BRCA pathway. DNA damage-inducible or S phase
specific monoubiquitination of FANCD2 requires the FA protein complex (A/C/G/E/F
complex). Monoubiquitination targets D2 to DNA repair foci containing BRCA1,
BRCA2, and RAD51. The BRCA2 protein may function upstream in this pathway, by
promoting D2 monoubiquitination, and/or downstream in the pathway by promoting
homologous recombination repair. (B) Whole cell lysates were prepared from the
indicated EBV lymphoblast lines (lanes 2-11) or BRCA2(-/-) CAPAN-1 cells (lane 12),
and cellular proteins were immunoblotted with a monoclonal antibody specific for
FANCD2 (F17 monoclonal). These cell lines and their growth requirements have
previously been described (5).
Supplementary References
1. R. M. Myers et al., in Methods Enzymology, R. Wu, Ed. (Academic Press, San Diego,
1987), vol. 155, pp. 501-527.
2. M. Whitney et al., Nature Genet. 11, 341 (1995).
3. A. P. Cuthbert et al., Cytogenet. Cell Genet. 71, 68 (1995).
4. C. Timmers, Mol. Cell 7, 241 (2001).
5. I. Garcia-Higuera et al., Mol. Cell 7, 249 (2001).
table S1. FA patients with Biallelic Mutations in BRCA2
Clinical history
Cell line
FA Subtype
Assignment
Age
(sex)
Abnormal
Pigmentation
Abnormal
Thumb
Bone Chromosome
Marrow
Breakage
Failure
Cancer
Mutant
Allele #1
(exon)
BIC
entry
Mutant
Allele #2
(exon)
BIC
entry
HSC62
D1
30 yr old
(M)
-
+
-
+
-
IVS19-1
G to A
(20)
-
IVS19-1†
G to A
(20)
-
EUFA423
D1
3 yr old
(F)
+
+
-
++
Brain
tumor
7691 insAT
(15)
-
9900 insA
(27)
4
HSC230
B
2 yr old
(M)
+
+
+
++
-
3033 del
AAAC
(11)
many
10204
A to T‡
(27)
many
EUFA579
U/A*
2 yr old
+
+
+
++
AML
7235 G to A
(13)
1
5837
TC to AG
(11)
1
+
+
+
++
AML
8415 G to T
(18)
2
8732 C to A
(20)
1
(F)
AP37P
U/A*
2 yr old
(M)
* U/A, unassigned FA subtype
† Family History of Cansanguinity
‡ Polymorphic STOP variant (ter3326)
BIC, Breast Cancer Information Core (www.nhgri.nih.gov/intramural_research/lab_transfer/bic)