Exon Primer name Sequence Amplicon size

Transcription

1 Supplementary Material PCR amplification of the BRCA2 gene The components of the PCR reaction were: 20mM Tris-HCl(pH8.4), 50mM KCl, 1.5mM MgCl 2, 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 Sequence Amplicon size 2 2-F GTT CCA GGA GAT GGG ACT GAA 348 bp 2-R ACA CAT AAG GAA CAG TTT ATG GTT 3 3-F CCA TAG TCA AGA TCT TTA GCA 466 bp 3-R ACT GAT TTG CCC AGC ATG ACA 4 4-F TTA CAA CTC CCT ATA CAT TCT CA 454 bp 4-R AAC CAG CCA ATT CAA CAT CAC A 5/6 5/6-F ATA TCT AAA AGT AGT ATT CCA ACA A 421 bp 5/6-R AAT TGC CTG TAT GAG GCA GAA T 7 7-F GTT ATA CCT TTG CCC TGA GAT T 398 bp 7-R GTC AGT TAC TAA CAC ACT TAT CA 8 8-F GTT TAT TCA CTG TGT TGA TTG AC 372 bp 8-R CAT ATA GGA CCA GGT TTA GAG A 9 9-F CAT CAC ACT ACT CAG GAT GAC A 495 bp 9-R GCA TGG TGG TGC ATG CTT GTA 10a 10a-F CCA AGT ACT CAG AAT AAC CCT T 497 bp 10a-R TTT GTC ACT TCC ACT CTC AAA G 10b 10b-F TCC ATG AAG CAA ACG CTG ATG A 470 bp 10b-R CCA GAT ATT GCC TGC TTT ACT G 10c 10c-F GAC CTA TTA GAC ACA GAG AAC A 454 bp 10c-R CTG CAT TCT TCA AAG CTA CAG A 10d 10d-F TCA GGT CAT ATG ACT GAT CCA A 416 bp 10d-R AAC ACA GAA GGA ATC GTC ATC T 10e 10e-F CCG AAA GAC CAA AAA TCA GAA CT 400 bp 10e-R AGC AAA CCA ACA TGG CAT ACG T 11a 11a-F CCA AAC ACT ACC TTT TTA ACT TAG T 380 bp 11a-R GAC CTC TTC TTT TAT ATC TGA AAC T 11b 11b-F CTG AAG AAC CAA CTT TGT CCT TA 350 bp 11b-R AGT GCT GGC ATT TTC ATG ATC AT 11c 11c-F TAT TAC CCC AGA AGC TGA TTC T 299 bp 11c-R TAC CTT TGA GCT TGT CTG ACA T 11d 11d-F ATG TCA CCC AGT ACA ACA TTC A 484 bp 11d-R CCT TTC ATT AGC AAC TTG GAA GA 11e 11e-F GAG AGT AGC ATC ACC TTC AAG A 464 bp

2 11e-R CTG CCC ATT TGT TCA TGT AAT C 11f 11f-F AAA CAA GCA ACC CAA GTG TCA A 433 bp 11f-R CAG AAA CAA CTA CAC TAC TCT GT 11g 11g-F GGA AAT CAA GCT CTC TGA ACA T 403 bp 11g-R CAT CTG GTT TTC AGG CAC TTC A 11h 11h-F CCC CTC AGA TGT TAT TTT CCA A 387 bp 11h-R ACC CCA CTT CAT TTT CAT CTG T 11i 11i-F GGA ATG CAG AGA TGC TGA TCT 346 bp 11i-R CAT TGA AAC GAC AGA ATC ATG AC 11j 11j-F CTG CTC ATG GCA CAA AAC TGA 331 bp 11j-R GAA TTT CTA CTG GCA GCA GTA T 11k 11k-F CTT CTG CAG AGG TAC ATC CAA 496 bp 11k-R TGC TCC GTT TTA GTA GCA GTT A 11l 11l-F CTG ATC AGC ACA ACA TAT GTC T 470 bp 11l-R CTT TTC ATC ACG TTC GGG TTG T 11m 11m-F GAT CAG AAA CCA GAA GAA TTG C 423 bp 11m-R ACA CTT TGG GGC AGC TGT GAT 11n 11n-F TGC AAA GGA ATC TTT GGA CAA AG 408 bp 11n-R GCT AAG GCT GAA TTT TCA ATG AC 11o 11o-F GTG GTG CCA CCT AAG CTC TTA 386 bp 11o-R GTA TCT TGT TTT TCG GAG AGA TG 11p 11p-F ACT TCT GTG AGT CAG ACT TCA T 371 bp 11p-R TGT GGG TAT GCA TTT GCA TCT T 11q 11q-F CAG TAG CAT GTC TAA CAG CTA T 348 bp 11q-R GTT TCA TGT GAA ACA CAA ACG AT 11r 11r-F GAT ATT TGC GTT GAG GAA CTT G 328 bp 11r-R GCG TGC TAC ATT CAT CAT TAT C 11s 11s-F GGA TAG CCA GTG GTA AAA TCG T 498 bp 11s-R AGA CTT GCT TGG TAC TAT CTT CT 11t 11t-F TCA CCT TGT GAT GTT AGT TTG GA 469 bp 11t-R CAT TTT GTC TAG ACG TAG GTG AA 11u 11u-F CTG CTA TAC GTA CTC CAG AAC A 431 bp 11u-R AAC AAG TGA GAC TTT GGT TCC TA 11v 11v-F GCA CTG TGT AAA CTC AGA AAT G 406 bp 11v-R TGT GGC ATG ACT TGG CAG TTT 11w 11w-F ACT TTT TCT GAT GTT CCT GTG AA 383 bp 11w-R TCC CCC AAA CTG ACT ACA CAA F CAT TTA AAG AGT CAA TAC TTT AGC T 332 bp 12-R GCA CAG TGG CTC ATG TCT GTA F GCA TCC GTT ACA TTC ACT GAA A 302 bp 13-R TAA CTT CTT AAC GTT AGT GTC ATT 14a 14a-F AGG CTA GCC TTG AAA AAT GTG A 495 bp 14a-R CAT CAG AGC GAT GTC CAT CAA 14b 14b-F TTG ATT ACT ACA GGC AGA CCA A 449 bp 14b-R TAA CAA GTC CAC AAG AAG TTA TC F TTT TGG TCA GGC TGG TCT TGA 482 bp 15-R TCA TTC ATC CAT TCC TGC ACT AA F TTT TGT AGT GAA GAT TCT AGT AGT 472 bp 16-R CAG AAT GCT TAA CCA TAA TGC AC

3 17 17-F CAC CAT GCT CAG CAA TGA AGT 498 bp 17-R GAT GGC AAC TGT CAC TGA CAA 18a 18a-F TCC ACT ATT TGG GGA TTG CTA A 432 bp 18a-R TAC CAC CCA TCT GTA AGT TCA A 18b 18b-F TAG AAG CAG AAG ATC GGC TAT A 389 bp 18b-R GAA TTT AAC TGA ATC AAT GAC TGA 18c 18c-F TCC TCC CCT CTT AGC TGT CTT 312 bp 18c-R GAC CTC CCA AAA ACT GCA CAA A F GGC AGT TCT AGA AGA ATG AAA AC 480 bp 19-R ACC CCT TCT CTA CCA AAA ATA CA F CTC AGG TGA TCC ACT AAT CTC 453 bp 20-R CCC TTG TTG CTA TTC TTT GTC T F TGA CAG AGT GAG ACC CTG TCT 411 bp 21-R CCT TTT GGA GAA ATG CAG CAT T F CAC ACC CTT AAG ATG AGC TCT 443 bp 22-R TAG TGG ATT TTG CTT CTC TGA TA F ATC CAC TAC TAA TGC CCA CAA A 416 bp 23-R TCC CGT GGC TGG TAA ATC TGA F ACA TAC AGT TAG CAG CGA CAA A 398 bp 24-R CAG ATC ACT AGT TAG CTA GCA A 25a 25a-F AGC TTT CGC CAA ATT CAG CTA T 361 bp 25a-R CTC TTG AAA GTG GCC CTC TTT 25b 25b-F GGA TAG ACC TTA ATG AGG ACA T 336 bp 25b-R TCC TGA GGT TCA TGG GCA ATT F GCA TGT TTG ACA ATT GGT ATC AC 427 bp 26-R GGA GCC ACA TAA CAA CCA CAT T 27a 27a-F GGG GAG GGA GAC TGT GTG TA 400 bp 27a-R TTT CGT ATT TGG TGC CAC AAC T 27b 27b-F AAG TCT TGT AAA GGG GAG AAA GA 379 bp 27b-R CTG GTG GGA GCA GTC CTA GT 27c 27c-F ATT CTC CTC AGA TGA CTC CAT T 352 bp 27c-R ACT GGA AAG GTT AAG CGT CAA T 27d 27d-F CTC AGA CTG AAA CGA CGT TGT A 318 bp 27d-R GCA ACT GAA GCA AAA GTA TAC CA 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

4 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 IC- B2-15.1R, and IC-B2-27.1F with IC-B2-27.1R, were obtained from Ingeny (Ingeny International, The Netherlands) ( 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).

5 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 mm 2 tissue culture plates. After 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 mm 2 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).

6 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 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 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).

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10 table S1. FA patients with Biallelic Mutations in BRCA2 Clinical history Cell line FA Subtype Assignment Age (sex) Abnormal Pigmentation Abnormal Thumb Bone Marrow Failure Chromosome Breakage 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) insa (27) 4 HSC230 B 2 yr old (M) del AAAC (11) many A to T (27) many EUFA579 U/A* 2 yr old (F) AML 7235 G to A (13) TC to AG (11) 1 AP37P U/A* 2 yr old (M) AML 8415 G to T (18) C to A (20) 1 * U/A, unassigned FA subtype Family History of Cansanguinity Polymorphic STOP variant (ter3326) BIC, Breast Cancer Information Core (