mutation analysis of five candidate genes in familial breast cancer

ORIGINAL PAPER

Mutation analysis of five candidate genes in familial breast cancer

A. Marsh Æ S. Healey Æ A. Lewis Æ A. B. Spurdle ÆM. A. Kedda Æ K. K. Khanna Æ kConFab ÆG. J. Mann Æ G. M. Pupo Æ S. R. Lakhani ÆG. Chenevix-Trench

Received: 8 November 2006 / Accepted: 16 November 2006 / Published online: 23 December 2006� Springer Science+Business Media B.V. 2006

Abstract Most of the known breast cancer suscepti-

bility genes (BRCA1, BRCA2, CHEK2 and ATM) are

involved in the damage response pathway. Other

members of this pathway are therefore good candi-

dates for additional breast cancer susceptibility genes.

ATR, along with ATM, plays a central role in DNA

damage recognition and Chk1 relays checkpoint sig-

nals from both ATR and ATM. PPP2R1B and

PPP2R5B code for subunits of protein phosphatase 2A

(PP2A), which regulates autophosphorylation of ATM.

In addition, EIF2S6/Int-6, which was originally identi-

fied as a common integration site for the mouse

mammary tumour virus in virally induced mouse

mammary tumours, is a candidate breast cancer

susceptibility gene because of its putative role in

maintaining chromosome stability. To investigate the

role of ATR, CHK1, PPP2R1B, PPP2R5B and

EIF2S6/Int-6, we carried out mutation analysis of these

genes in the index cases from non-BRCA1/BRCA2

breast cancer families. We also screened sporadic

breast tumours for somatic mutations in PPP2R1B

and PPP2R5B. Although we identified many novel

variants, we found no evidence that highly penetrant

germline mutations in these five genes contribute to

familial breast cancer susceptibility.

Keywords ATR � BRCA1/2 � CHK1 � EIF2S6/Int-6 �Familial breast cancer � PPP2R1B � PPP2R5B

Introduction

Approximately 5% of all breast cancer occurs in mul-

tiple-case families, but mutations in the known high-

risk breast cancer susceptibility genes can only be

identified in about a third of these families [1]. The

failure to identify additional breast cancer susceptibil-

ity genes by linkage analysis suggests that no one gene

accounts for a substantial amount of the familial

aggregation in non-BRCA1, non-BRCA2 breast cancer

families [2]. The majority of breast cancer families may

therefore be due to rare, highly penetrant mutations in

many different genes, or be polygenic in nature [3].

Candidate high-risk genes include those whose protein

products function in cellular pathways that are known

to be associated with breast cancer. The DNA damage

response pathway is an example of such a pathway,

where inherited mutations in several genes (BRCA1,

BRCA2, p53, CHK2 and ATM) are linked with breast

cancer predisposition [4–6].

A. Marsh � S. Healey � A. Lewis � A. B. Spurdle �K. K. Khanna � S. R. Lakhani � G. Chenevix-Trench (&)Cancer and Cell Biology, Queensland Institute of MedicalResearch, c/o RBH Post Office, Herston, Brisbane, QLD4029 , Australiae-mail: [email protected]

M. A. KeddaQueensland University of Technology, Brisbane, Australia

kConFabThe Kathleen Cunningham Foundation for Research intoFamilial Breast Cancer (kConFab), Peter MacCallumCancer Centre, Melbourne, Australia

G. J. Mann � G. M. PupoWestmead Institute for Cancer Research, Sydney, Australia

S. R. LakhaniMolecular and Cellular Pathology, School of Medicine,University of Queensland, Brisbane, Australia

123

Breast Cancer Res Treat (2007) 105:377–389

DOI 10.1007/s10549-006-9461-z

One candidate gene for breast cancer susceptibility

is that encoding the BRCA1-interactor, ATR. ATR

belongs to the PI3/PI4-kinase family, and is closely

related to ATM [7]. ATR and ATM share similarity

with Schizosaccharomyces pombe rad3, a cell cycle

checkpoint gene, which arrests the cell division cycle in

response to DNA damage [7]. In mammals, ATR, in

conjunction with ATM, plays a central role in DNA

damage recognition to initiate checkpoint responses to

DNA damage. ATR mediates responses to a broad

spectrum of genotoxic stimuli whereas ATM has

evolved selectively to deal with ionizing radiation and

other radiomimetic agents that cause double strand

breaks in DNA. ATR has been shown to phosphory-

late checkpoint kinase CHK1, checkpoint proteins

RAD17 and RAD9, as well as BRCA1 [8]. Normal

activity of ATR and the pathways it is involved in are

vital for normal cell functioning. Biallelic disruption of

ATR in mice leads to early embryonic lethality [9, 10].

However, heterozygous (ATR+/–) mice display only a

small decrease in survival, but do exhibit an increase in

tumour incidence [9]. The link between tumorigenesis

and ATR has also been observed in humans, as somatic

ATR mutations have been found in sporadic stomach,

lung and breast tumours [11, 12]. This link is further

supported by expression studies conducted by the

Cancer Genome Anatomy Project, which have shown

reduced expression of ATR in breast tumours com-

pared to normal myoepithelial cells, suggesting that

reduced expression of ATR might promote tumori-

genesis [13, 14]. Although extremely rare, homozygous

mutations in the ATR gene in humans cause Seckel

syndrome, which shares several features in common

with ataxia telangiectasia, including cancer suscepti-

bility [15, 16].

Chk1 protein kinase is a key component of the DNA

damage checkpoint response and therefore another

candidate for a breast cancer susceptibility gene. Chk1

relays checkpoint signals from ATR after activation

with ultraviolet light, stalled replication or certain

drugs and from ATM in response to ionising radiation

[17, 18]. BRCA1 is also required for optimal activation

of Chk1 after DNA damage [19]. There is evidence to

suggest that Chk1 heterozygosity can contribute to

tumorigenesis by allowing inappropriate entry into S

phase, accumulation of DNA damage and failure to

prevent entry into mitosis after damage [20]. Two

mutations of CHK1 of unknown significance have been

reported in primary breast tumours [21] and Chk1

over-expression in the human breast cancer cell line,

MCF-7, inhibits cell proliferation [22].

Protein phosphorylation is a regulatory mechanism

commonly employed in many cellular processes

including cell cycle progression, growth factor signal-

ling and cell transformation. Protein phosphatase 2

(formerly 2A), regulatory subunit A (PR 65), beta

isoform (PPP2R1B) and protein phosphatase 2, regu-

latory subunit B (B56), beta isoform (PPP2R5B) code

for subunits of protein phosphatase 2A (PP2A). PP2A

regulates the magnitude of ATM autophosphoryla-

tion—on serine 1981 [23]. The scaffolding (A) and

catalytic (C) subunits of PP2A interact with ATM in

undamaged cells and ionizing radiation induces phos-

phorylation-dependent dissociation of PP2A from

ATM and loss of the associated protein phosphatase

activity. It therefore appears that PP2A plays an

important role in the regulation of ATM autophos-

phorylation and activity in vivo. Mutations in

PPP2R1B have been detected in about 15% of lung

and colorectal tumours [24], and these mutations

interfere with the binding of regulatory subunits [25].

Recently Esplin et al. [26] demonstrated that the fre-

quency of the glycine 90 to aspartic acid (G90D)

polymorphism in PPP2R1B is associated with a low

but statistically significant frequency of inherited

breast cancer and that the role of this alteration in

transformation is likely to involve decreased interac-

tion with the B56c subunit.

Although not known to be directly involved in the

DNA damage response pathway, EIF3S6/Int-6 is an-

other breast cancer susceptibility gene. Int-6 was orig-

inally identified as a common integration site for the

mouse mammary tumour virus (MMTV) in virally in-

duced mouse mammary tumours. This insertional

event creates a dominant negative C-terminally trun-

cated protein and leads to breast tumour formation in

mice. Consistent with this, expression of a C-terminal

truncated EIF3S6/Int-6 gene in human mammary epi-

thelial cell lines leads to transformed phenotype [27].

EIF3S6/Int-6, located at 8q22-23, encodes the p48

component of the eukaryotic translation initiation

factor-3 (eIF3-p48), and plays a pivotal role in the

initiation phase of protein synthesis [28, 29]. Human

EIF3S6/Int-6 has been implicated in mitotic progres-

sion, as HeLa cells depleted in EIF3S6/Int-6 by RNAi

show defects in spindle formation, chromosome seg-

regation and cytokinesis [30]. These findings suggest

that reduced expression of the EIF3S6/Int-6 gene in-

duces oncogenic properties associated with chromo-

somal instability. Miyazaki et al. [29] surveyed 100

primary sporadic breast tumour DNAs and found loss

of heterozygosity (LOH) at EIF3S6/Int-6 in 11 of 39

(28%), although no mutations were observed in

the gene. In addition, expression studies by the

Cancer Genome Anatomy Project have shown

reduced expression of EIF3S6/Int-6 in breast tumours

378 Breast Cancer Res Treat (2007) 105:377–389

123

compared to normal myoepithelial cells, suggesting

that EIF3S6/Int-6 might have some tumour suppressor

function [13, 14].

The aims of this study were to determine whether

ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6

are breast cancer susceptibility genes. We also

screened 43 sporadic breast tumours for mutations in

PPP2R1B and PPP2R5B because mutations in

PPP2R1B have been detected in 5/33 (15%) primary

lung tumours, 4/70 (6%) cell lines derived from lung

tumours and 2/13 (15%) primary colorectal tumours

[24] but breast tumours have not been previously

screened for somatic mutations in these genes.

Materials and methods

Multiple-case breast cancer families

Multiple-case breast cancer families were ascertained

through the Kathleen Cuningham Foundation Con-

sortium for Research into Familial Breast Cancer

(kConFab) [31]. The ascertainment criteria for families

without mutations in BRCA1 or BRCA2 have been

described previously [32]. Briefly, families containing

only female but not male breast cancer cases, which

had undergone full sequencing or DHPLC analysis of

BRCA1 and BRCA2, were selected for mutation

screening. Index cases were defined as the youngest

available breast cancer case. Mutation analysis was

performed for ATR (65 index cases), CHK1 (58 index

cases), PPP2R1B (55 index cases), PPP2R5B (55 index

cases) and EIF2S6/Int-6 (67 index cases), subject to

DNA availability. The same 55 familial cases were

analysed for mutations in all five genes.

A subset of kConFab families had been included in

a 10 cM genome-wide search for novel breast cancer

susceptibility genes in multiple-case breast cancer

families from which BRCA1 and BRCA2 mutations

had been excluded by high-sensitivity methods and in

which no haplotype was shared at either locus. The

index cases qualified for ATR, CHK1, PPP2R1B,

PPP2R5B or EIF3S6/Int-6 mutation analysis if an

individual family logarithm of the odds (LOD) score

under heterogeneity or a non-parametric LOD score of

‡0.5 had been obtained at any of the markers closest to

or flanking ATR (D3S1292, D3S1569 and D3S1279),

CHK1 (D11S908, D11S925, D11S4151 and D11S1320),

PPP2R1B (D11S898, D11S908 and D11S925),

PPP2R5B (D11S4191, D11S987 and D11S1314) or

EIF3S6/Int-6 (D8S270, D8S1784 and D8S514). In

addition to the 55 cases described above, index cases

from families showing haplotype sharing among all

affected in the kindred, around loci of interest, were

analysed. Two families showed haplotype-sharing

around ATR, 12 families around CHK1, 16 families

around PPP2R1B, 13 families around PPP2R5B and

four families around the EIF3S6/Int-6 gene. There

were two families (both of which carried ATM c.1066-

6T > G families (Families B and C) from [33]) which

showed haplotype sharing around CHK1, PPP2R1B

and PPP2R5B, all of which are located on chromo-

some 11 on which ATM is also located. Forty-three

sporadic breast tumours were obtained from the Peter

MacCallum Cancer Centre Tissue Bank. These sam-

ples were from individuals with no family history of

breast cancer and had not been pre-screened for

mutations in BRCA1 or BRCA2. Matching germline

DNA was subsequently requested from all tumours

with PPP2R1B or PPP2R5B variants that were not

reported as SNPs in the databases.

Forty-six DNAs from additional family members of

the index case identified with potential ATR patho-

genic variants were also genotyped, as well as DNA

extracted from one archival tumour section from a

deceased female diagnosed with breast cancer. An

additional 336 non-BRCA1/BRCA2 index cases from

kConFab were screened for putative ATR missense or

splicing variants identified by mutation screening.

Controls

Controls were females with a mean age of 46.2 years

(range of 16–72 years of age) with no reported per-

sonal or family history of cancer, recruited through the

Australian Red Cross Blood Service. Each control

gave informed consent to provide a sample of blood for

molecular studies, and complete a short questionnaire

assessing risk factor information. All samples and data

were de-identified. Ethics approval for this study was

obtained from the Australian Red Cross Blood Ser-

vices, the Queensland University of Technology and

the Queensland Institute of Medical Research. DNA

was extracted from blood aliquots using the salting out

procedure [34] and lymphoblastoid cell lines (LCLs)

were generated using standard EBV-transformation of

Ficoll-separated lymphocytes.

DHPLC analysis

Primers were designed using the web-based program,

Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/pri-

mer3_www.cgi), to cover the 47 exons of ATR (Gen-

bank: NT_005612), 12 coding exons of CHK1

Breast Cancer Res Treat (2007) 105:377–389 379

123

(GenBank NM001274), 16 exons of PPP2R1B (Gen-

bank: NT_033899) and 13 exons of PPP2R5B (Gen-

bank: NT_033903) and 13 exons of the EIF3S6/Int-6

gene (Genbank: NT_008046) (Table 1). PCR products

were amplified from 15 ng DNA using AmpliTaq Gold

(PE Applied Biosystems, Forest City, CA, USA) and

16–20 pmol of each primer (Table 1) in a 20 ll final

volume. Cycling conditions for all fragments were 94�C

Table 1 CHK1, PPP2R1B, PPP2R5B, ATR and EIF3S6 primers and DHPLC conditions

Gene-exon Forward primer Reverse primer Ampliconsize (bp)

DHPLCTemp(�C)

CHK1-2 TAGAAGGGGAAGGCAAGAGC CTCAGAAAACGAAGGCAAGC 237 61CHK1-3a AGCAGAAACCACTTCCTTGG TCCATACCTATTCTGTCAAAAAGC 367 55CHK1-3b TTCTATGGTCACAGGAGAGAAGG TCCAATTTCACAGTTGCATGA 200 54CHK1-

4&5GAAGCTATGTGGTTGCTACCTG GACTTGATTTTGCCTTGTATGG 397 56, 51

CHK1-6 TTGCAAAACATTTTTATTCAGTGTC TGACTTTTTATAGGAGTTTTACCATGA 277 57, 52CHK1-7 CTGCCATGCCTATCCTGATT TCCAAGAAATCGGTACTCTTTCA 331 56, 51CHK1-8 CCTCAAGCCATAGGCTTCTC GCCTGCCTAGCTTCCCTTTA 447 55, 50CHK1-9 GCATAGAAGACTTGAAAGCATTTG CAGGCCTTTCTTATATCACACACA 292 57CHK1-10 AAGCATGAGAACTTGTGTGTGA AAATAAAGAGCTGCCATTACTTTTA 308 58, 53CHK1-11 TGGATTTATTCATTTGTCTTCTGTTT AGGTGTGAGCCACAGCCTAT 272 56, 51CHK1-12 CACCCATGTGGCTTAACCTT CAAGTAACCTATTTACAAATGCCACA 243 53CHK1-13 GACCGAAAAGAAAATGGTAGC TTCTATTCATCCTTTCCCCAAA 455 58, 53PPP2R

1B-2ACCAGCAGCAGGAGGAGAA CCTTCCCCCTTCTCTACCAC 203 62

PPP2R1B-3

GGCTAGTGAGGCCGAGTTAC ACTCTGTTCTGTTTCTTTTGCAC 413 56

PPP2R1B-4

GTGTCAGTGTCCAGCTGTGG TGCTGCAGTGTGTCTATCACC 249 59

PPP2R1B-5

ACGTGTACCACCTCCCTTCA CCTAGTATCAGCCACTCATTGGT 465 60

PPP2R1B-6

AAAACCTCTAAAAGTCTGTCTGTGTG TTAACCAGGCCAGAATCAGC 370 58

PPP2R1B-7, 8

GTTCCCCATGGGTCTCAGTA TGCACCAAAATGAATGACACA 573 54, 59

PPP2R1B-9

TTGATAAACCTGGTTAAATGTCCTC TCAAAGAGCTTTAAGGTAAACAAACA 249 54

PPP2R1B-10

TGGTATGGAAGTTTCTGTTGTCTT CATTTTCTTTTTGGGGGTTC 292 54

PPP2R1B-11

CCCTGACAGGAGACTGTTGG AACAAGGCCATACTTTCATGC 370 60

PPP2R1B-12

GCCTTCAGGTAACCTTGGTG TCTGGTCAGACTGAGATTCCTTT 239 55

PPP2R1B-13

TGCTTTTGGTCCACATTTGA TTCTCTCATCTCCTGTCTATTCCA 394 52, 56

PPP2R1B-14

TGTTTCATTAGATTTTGGAATTTTT AGCTAATTACTCCCCACTATAAGGT 280 51, 56

PPP2R1B-15

CCATTAACCATATACACCAAAAAGG CCCTCCCTGTTTCCCATAAT 285 56

PPP2R1B-16

CACAACTGAGGGCATCCTG CAGCTCCCTGCACACCAT 297 62

PPP2R1B-17

GTCACCGGGAATGTGAAAGT GCTTCATCACCCTGTAAACCA 291 55, 60

PPP2R5B-2A

AAAGACCTGGGCACTCCAC AGGGGGCAGCTTCGTCTC 389 62

PPP2R5B-2B

CAGTCTCACCCAGCACCTC CCTGGAGGGGTATCCAAACT 392 64

PPP2R5B-3

TGGAGATTAGGAGGGTCTGC GCCAAGCCGATGAGTACAG 392 58, 63

PPP2R5B-4

GAGAGACTCCCTGGCTTCTG GGCCCTCATCCAACTGTCTA 263 60

PPP2R5B-5, 6

CCATAGGGTGGAATGAGTGG TTTGCCGCAGTCTTCTAACA 496 62


123

Table 1 continued


DHPLCTemp(�C)

PPP2R5B-7, 8

CCTACCCTGCTTGCTTTGAG ACAGGCCTGGGCCTCAC 329 62

PPP2R5B-9, 10, 11

GTGCCTTCCTGACCTGTCTT CAGCTACCCAACCTTTCCAG 540 62

PPP2R5B-12

GAGGTAGTGATGGACACCAGA TGGTCCCAGTTTCTGTGAGTT 250 60

PPP2R5B-13

TATGTGAAGGGCAGGGAAAG CAGACAGAGTTTGGGGTAGTCC 280 56, 61

PPP2R5B-14A

GGACTGTGAGGGAGTCTGGA CAGCTGGAGCCTAGCTTTG 500 62

PPP2R5B-14B

AGGACTTGACAAGGGCAAGC TGGCCCTTCTAAACCAATCTT 498 57, 62

ATR-1 AGCGGTAGCTTTGGAGACG GAGAGCACGTGAAACCCAAG 194 63ATR-2 TTTTGCAAATGAAACTGAGTCAC TCCATATCTAAAACTACATGGAGAAAA 250 54ATR-3 CAGGGGTTACAGGAGTGAGG GATGGCAAGTGAATGCATGA 284 58ATR-4A TCAATCGTCAAGGATTTAGCAA GAACACAACCTATCTGCCAAAG 494 56ATR-4B AGCTCCTTTGCAGTTGATGAG GAGCACACCGTCTTCAAACA 430 56ATR-4C TTTCCCTTTGAAAGCAGAAGC TCCATAATAGTGAGCCAGACTACA 426 52, 56ATR-5 AATACATTCTTGCTGCCTATGAA GCACTCCCTTGGCTACATTT 390 52, 56ATR-6 TCATGTTTTAACCAAATTTATATTGCAT CCAACCCCAAGAAACAAGAA 284 50, 56ATR-7 TTTCGTACTCACAATTTCTTTTGG ACACGCACTTAGGCTTCAGG 385 55ATR-8 TCACAGACCTCAGACCATCG CACACACACATTCTTGTGAGCA 336 50, 55ATR-9 CCTTTTTCAGTGCACTTGCTT AACCCTGCATACATAGCCAGA 465 52, 57ATR-10 TTTTGTCCCAAATTAAGCAAGAC CCTGTAATTTTTCAAGGCTTCAG 440 56ATR-11/12 GAACTTAGGATTCTTCATGGCATA AGAACACAAATGCTGCCAAG 505 54ATR-13 TTTCCGTTTGTTTCTGGGATA CAAACCTCTACATGCTGCAA 433 52, 57ATR-14 CTGCCCTCTATGGTGGCTTA CTGAATTGCAAACCTCAAGTG 295 58ATR-15 GACTCCAAATATGTGTGGCATTT GGCTATAATTTACTCACCCTCTTTCC 402 50, 55ATR-16 TGGGCTAGAGCTCCTGATGT AATGGCTAGCAGCCAGAAAA 400 56ATR-17 TGTGAAGATATATGCTTTTGGAGAA TTTCTCATCTTTGAATGTTTGTAGC 350 53ATR-18 TGTCCTTAGGGCTCATCTGC AATAAACTCAGGCAGTCATTTACC 226 52, 58ATR-19 TCCATGTTTAAAGCTGCCTTTT TGACAGTTTCCACATTACCATCA 363 52, 56ATR-20 GCAGCTCAGTAATCAACTAGCA TCCCTCCAAAAGTCATGGAT 396 52ATR-21 GAGAATTCAGGCCTTTGGAA TTCAAGCTCAAAGGGCATCT 377 51, 56ATR-22 TGTGAACTCATCAAAAACTAGCTGA CACCCTAGATGGGGTTCAAA 571 52, 56ATR-23 CCATGGAAAAAGCAGTACACC AATTAAACAATTTAAAGCCAGTAATGA 233 51, 56ATR-24 GCGTTATCTGCTTCACTGTGG GATTACAGGCGTGAGCCACT 265 53, 58ATR-25 AGTCAACTGAAGGAGTTGCTG TTGTGTGTGCTAGGCATTCAG 289 55ATR-26 TTATCTCACATGCTACTCTTTGACA CATTTCCTACTAATAGGTAGCCTTTC 308 55ATR-27 AATGGTTAGCTTTAGATGTCATA AAAGAAACAGAAGTGATAACTCAC 315 52, 58ATR-28 GGCAGAGTTGGAGCAAGACT AAGACTGGCCACATTAATTTTCA 412 52, 56ATR-29 CAGGTGGTTTTATAGTTTTATTTCCAG CCAGAGTTCCTATTCAGGGCTA 296 53, 58ATR-30 AAGGGCAATAAGGTAAATAGTAAT AAATTACCCAATTCACTAACTAAAAA 279 54ATR-31 ATGAAAAATCTCTGAATGTCACC ACCGCACCCATCCTAAAACT 204 51, 56ATR-32 GACCCAGCTACCGTGACATT TTTTCCAATCCTGTCAGGTG 286 56ATR-33 TTAAAGTTCTACCAAAGACAACTGTG CAAACACCCCCAAATAATATCC 382 52, 58ATR-34 ATTGGGAACAGAGGCTTTCA GACATTTCCCTGGCCATTAC 313 55, 60ATR-35 AAAGGCTTATTGTTGCCTTTTT TGATGTGGTGGCAGTTTCAT 387 56ATR-36 TCACATACTTTTGATCCCTAATCA ACCTAGAATATGCTAAGACATGTGA 330 52, 57ATR-37 TTTTTGTGAAAACGGTATGTGG AGACTGTCCAGCCAAATCTGA 280 54ATR-38 TAACCTTGGGCATGGGAATA GAGACGCCCTGGAACTTGTA 463 56ATR-39 GCTCTTGATAGCATTGCCAAA ACCCAACATCAGTTTATAAGCAC 331 54ATR-40 AAAACCACAGACTGCTGAACC TTTTTGCCATCAGTACAAATGAGT 486 50, 56ATR-41 CACAGAAATTTTTGGCCCTA GCAACTCTGAAATAAAAGCAATC 288 54ATR-42 TTTGGTTATGAAATGAACAATCTTT AGGAAGGGATGGAAACACTT 410 48, 54ATR-43 AGTAGATGTTTCTTGTCCAATTTTAAC CATATGAGGCCAATATAAATCTAAAA 328 52, 56ATR-44 GTTGTTATGGTTGAATGTTTATTTTTA CAAGGAAGATACAGTTGTTGAGAA 320 50, 56ATR-45 GCAAAGGCAGAGCTACATGG CAAACATATGTAGGGGCCAAT 397 52, 57ATR-46 CCATTAGCTTCTCATCCTTCA TTTCCAAGCAGTTCTCATGC 305 56ATR-47 GGGGTATTGGTCAGTAAAATGG GAGAAATAACAGTTGCTGAGAACG 412 51, 56


123

for 12 min, followed by four sets of four cycles of 94�C

for 30 s, 61–55�C for 45 s and 72�C for 30 s, with the

annealing temperature dropping 2�C after each set of

four cycles, followed by 30 cycles of 94�C for 30 s, 55�C

for 45 s and 72�C for 30 s, and a final extension of 72�C

for 7 min. The amplification of fragments was opti-

mised as needed by adjusting the MgCl2 concentration,

adding 0.5 or 1 M Betaine, or by lowering the annealing

temperature. Amplicons were denatured at 95�C for

5 min and re-annealed slowly by gradual cooling to

60�C over 30 min prior to injection onto the Varian

Helix System (Varian, Walnut Creek, CA, USA).

DHPLC was carried out at the recommended melt

temperature for each exon (Table 1) as determined by

the DHPLC melt program (http://insertion.stanford.

edu/melt.html). DHPLC of EIF3S6/Int-6 was also

carried out at 2�C above the recommended tempera-

ture, as this increases sensitivity to >99% [35–37].

DHPLC data was analysed using the Star Workstation

version 5 (Varian). Samples with a heterozygous peak

or an aberrant shift in retention time and/or peak shape

were re-amplified for sequencing. DNA sequencing was

performed with both forward and reverse primers using

the ABI Prism Big Dye Terminator cycle Sequencing

Ready reaction kit (PE Applied Biosystems) and

analysed on an ABI 377 sequencer.

Variant analysis

Rare variants identified by sequencing were analysed

further to give an indication of their functional signif-

icance. Coding variants and variants located near the

exon/intron boundary were analysed in silico for amino

acid changes, predicted splicing defects (using BDGP

Splice Site Prediction (http://www.fruitfly.org/seq_

tools/splice.html), SpliceSiteFinder (http://www.genet.

sickkids.on.ca/~ali/splicesitefinder.html) and ESE Fin-

der (http://rulai.cshl.edu/tools/ESE/)), and predicted

mRNA folding changes (mFOLD (http://www.bioinfo.

rpi.edu/applications/mfold/old/rna/)). Blood donor

controls were screened by DHPLC, using the same

primers and conditions as described above, to deter-

mine the frequency of the variants in the general

population. Bayesian causality analysis was conducted

on families carrying any rare missense or splicing

variants that were not found in controls [38].

RT-PCR

To investigate the effect of the predicted donor splice

site change, CHK1 c.354 G > A, lymphoblast cell lines

were cultured from one index case carrier and two wild-

type blood donor controls. Total RNA was extracted

Table 1 continued


DHPLCTemp(�C)

ATR-11LOH

TTGTATACTCTTGTCCTTCAGCTTTC CTTCAGAGTCCAAGGATTCCA 270 N/A

ATR-14LOH

CAGATGACAGCACTTCCGAAT CTGAATTGCAAACCTCAAGTG 195 N/A

ATR-11SPLICE

CTGAACACGGACATGTGGAC CTTTTAGCTGCAACCAGAGC 559 N/A

ATR-14SPLICE

CCGCAAAAGGAGATTTGGTA TCCTAAAGTTCGAATGAGAGCA 419 N/A

ATR-27SPLICE

TTTGCAGAATGGTCAGCATC CAAGCCAAGGCTTTCATGTT 684 N/A

ATR-41SPLICE

AACCATGCTAGCCATGAACC CAGGGAATGTTCTCAGAAACC 502 N/A

EIF3S6-1 GTCGTTGTCCTCTGCTCTCC CTTGCTCAGCACCTCATCC 345 61EIF3S6-2 TCAGTTTATTTGGGGAACTTAAA GCCTTTTGGCTAAAGCTGTC 264 53EIF3S6-3 AACATTGTAGTGCTCTTCTGCATT TGGGATACTGGATAAAGTTTGAA 235 52, 57EIF3S6-4 TTTAAGGTGACTTTGCGTAGG TTAGGCCAAGCCATAAGCTC 235 54EIF3S6-5 GACTTATGAATTACAATGGGGTTT TTGAGTGGGAGGGTTTTCTTT 334 53EIF3S6-6 GGCTAGAGGGATACATGGGTA TCCTCATCAGAAAGTGTGAAGG 281 51, 56EIF3S6-7 TGTTTGTCCTCCTGCCTCTT CAGGTGACAACAAATTCAAATG 297 52, 56EIF3S6-8 TGACCTGCTCGAGAGTTTTCT AAAAGCCCATTTGAGGGTCT 360 50, 56EIF3S6-9 GGGATATTCTGGGGCAATCT GGAAGACACTGCAAGATTAACG 253 54EIF3S6-10 TGATTCACGTTTCAGGCAAT TTGTTTCATTGTTCTTCTGAAAGC 311 51, 56EIF3S6-11 CCAAAAAGTATCTATGCAGAGTGTG TTCCCAAAAGTATAATCAGGAACTG 259 54EIF3S6-12 GCGCAAAGGTATGTCATGCT TGTAACAATTACATGGTTGCTAAAC 277 56EIF3S6-13 TTCTTTTCACGGTGTTGATTTC CGTGATAAAATGGTAGGGAGCTA 445 54


123

from cell lines using Tri-reagent (Sigma) following the

manufacturer’s instructions. cDNA synthesis was

primed with random hexamers and carried out in a

volume of 20 ll on 1 lg of total RNA using Superscript

III (Promega). Two microlitre of cDNA was used to

amplify products designed to cover the regions carrying

the predicted CHK1 exon 4 splicing change. Standard

PCR conditions were used as described above with

8 pmol of primers forward 5¢-TGCCGTAGACTGTC-

CAGAAA-3¢ (CHK1 · 4RTF) and reverse 5¢-CC-

CTTTCATCCAACAGAAGA-3¢ (CHK1 · 4RTR).

The RT-PCR products, covering a region including the

Table 2 ATR variants identified in non-BRCA1/2 cases

Nucleotide changea Protein effect No. of heterozygousfamilial breastcancer cases

Controls SNPperb Likely pathogenicity

c.632 T > C p.M211T 35/67 Nottested

rs2227928 No—common SNP

c.946 G > A p.V316I 8/67 Nottested

No No—common SNP

c.1170 + 74 TAAins

4/67 Nottested

No No—common SNP and deeplyintronic

c.1326 A > G p.K442K 7/67 Nottested

No No—common SNP and silent

c.1776 T > A p.G592G 34/67 Nottested

rs2227930 No—common SNP

c.1886-60 A > C 1/67 Nottested

No Not likely—deeply intronic

c.1886-77 ins/del 1/67 Nottested


c.2437 A/G p.M813V; predictedto alter an ESE

1/67 0/175 No No—poor segregation in the familyand no evidence of spliceabnormality

c.2634-61 A > G 33/67 Nottested


c.2634-63 G > A 32/67 Nottested


c.2634-74 C > T 29/67 Nottested

rs9869842 No—common SNP and deeplyintronic

c.2875 G > A p.V959M 1/67 10/175 No No—common SNPc.3120 G > A p.L1040L 7/67 Not

testedNo No—common SNP and silent

c.3171 + 44 G > A 7/67 Nottested


c.3171 + 47 T > C 7/67 Nottested


c.3172-26 T > A 8/67 Nottested


c.3450-22 G > C 1/67 Nottested


c.3946-48 G > A 7/67 Nottested


c.4152 + 72 G > A 1/67 Nottested


c.4846 T > G p.S1616A 1/67 0/175 No No—poor segregation withcancer in families

c.5208 T > C p.Y1736Y 31/67 Nottested

rs13070715 No—common SNP and silent

c.5739-15 del 9 7/67 Nottested

No No—common SNP and intronic

c.7041 + 8 G > A Predicted to affectsplicing

1/67 0/175 No No—poor segregation withcancer in families

c.7875 G > A p.Q2625Q 14/67 Nottested

rs1802904 No—common SNP and silent

a Genbank Accession No. NT_005612b Variants have been previously reported in the SNP database (http://snpper.chip.org/bio/)


123

3¢ end of exon 3, exon 4 and exon 5, were visualised on a

1.5% agarose gel. Defects in the normal splicing of the

ATR transcript were also analysed in carriers of vari-

ants located near splice junctions, or predicted to affect

ESEs. Evidence for splicing defects was sought by the

analysis of the bands seen on a 1.5% agarose gel.

Loss of heterozygosity (LOH) analysis at the ATR

locus

Archival paraffin embedded tumour blocks were

available for some family members for LOH analysis.

One haematoxylin and eosin-stained slide was reviewed

by a pathologist to identify regions with at least 70%

tumour component, and DNA was isolated from the

tumour cells scraped from an adjacent section [39].

PCR and sequencing analysis was conducted on the

region containing the relevant variant as described

above. In some cases we were unable to amplify the

tumour DNA using the original primer pair, so primers

were re-designed to reduce the size of the PCR product

(Table 1). The height of the wild-type and mutant

peaks of the tumour DNA were compared to the

germline DNA in order to measure whether LOH had

occurred, and if so, of which allele.

Results

We identified a total of 24 ATR sequence alterations

in the index cases of non-BRCA1/BRCA2 families

(Table 2), including four potentially pathogenic vari-

ants, c.2437 A > G (p.M813V), c.2875 G > A

(p.V959M), c.4846 T > G (p.S1616A) and c.7041 + 8

G > A. We then screened 175 controls for these four

variants, and found that c.2875 G > A (p.V959M) was

quite common. For the remaining three, we carried out

LOH analysis in five breast tumours from carriers and

showed that no LOH had occurred (data not shown).

The c.2437 A > G (p.M813V) variant did not seg-

regate with breast cancer, as the variant was found to

be inherited from the father of the index case who had

no personal or family history of breast cancer, and not

from the affected mother with a strong family history.

Despite the predicted ESE change, RT-PCR analysis

of LCLs from a c.2437 A > G (p.M813V) carrier

showed no evidence of aberrant splice products (data

not shown). The c.4846 T > G (p.S1616A) ATR variant

showed partial segregation with breast cancer. The

index case, her mother and her aunt, all diagnosed with

breast cancer, were carriers of the variant, but the

cousin of the index case, diagnosed with breast cancer

at age 53, was not a carrier. The c.7041 + 8 G > A

variant was carried by both the index case and her

affected sister. In addition, genotyping of the archival

tumour block from the affected mother of the index

case showed that she too carried the c.7041 + 8 G > A

variant. We screened a further 336 non-BRCA1/

BRCA2 index cases from kConFab for the c.4846

T > G (p.S1616A) and 7041 + 8 G > A variants and

identified them in one other breast cancer family each.

However, genotyping of five and 12 additional mem-

bers of these two families (including three affected

women), respectively showed that the variants were

only carried by the index case, and not any other family

members.

We then conducted Bayes factor analysis [38] on all

the families carrying the c.2437 A > G (p.M813V),

c.4846 T > G (p.S1616A) and c.7041 + 8 G > A vari-

ants, assuming a standard model of autosomal domi-

nant breast cancer susceptibility used in a variety of

linkage studies and based on the segregation analysis

of Claus et al. [40]. One family carrying the c.4846

T > G (p.S1616A) variant showed close segregation

with breast cancer (odds in favour of causality of 7.8:1),

but the other family did not (odds against causality of

1.8:1), resulting in an overall odds for the c.4846 T > G

(p.S1616A) variant of 4.3:1 in favour of causality.

Analysis of the single family carrying the c.2437 A > G

(p.M813V) variant showed evidence against this vari-

ant being a high-penetrance breast cancer susceptibil-

ity allele (odds against causality of 6.8:1), while the two

families with the c.7041 + 8 G > A variant were only

minimally informative and gave combined evidence for

this variant of 1.5:1 against it being disease-associated.

We detected eight CHK1 sequence variants within

the non-BRCA1/BRCA2 individuals screened by

DHPLC, four of which were common and/or previ-

ously reported in the SNP databases (Table 3). Two of

the rare CHK1 variants were deeply intronic altera-

tions and so not investigated further but we did

examine the remaining two rare variants in controls

and in silico. These analyses predicted that c.354G > A

might disrupt the donor splice site at the end of exon 4.

However, we did not detect any splice variants in LCL

RNA from the individual carrying this variant (data

not shown). We also predicted that the c.718 + 10delT

intronic variant might introduce an exonic splicing

enhancer motif recognised by the human SR protein,

SRp55, within 10 bp of the exon/intron boundary that

may affect splicing but no LCL was available to

investigate this.

We identified seven different sequence variants

in PPP2R1B in non-BRCA1/BRCA2 index cases


123

Table 3 CHK1 variants identified in non-BRCA1/2 cases

Nucleotide changea Protein effect No. of heterozygousfamilial breastcancer cases


c.66-36 G > T 23/70 Not tested rs491528 No—deeply intronic and a common SNPc.354 G > A p.V118V 1/70 0/84 No No—silent and aberrant splice variants

not detected in LCLc.424 + 74 T > C 1/70 Not tested No Not likely—deeply intronicc.718 + 10delT 1/70 0/86 No Not likely—intronic and no LCL

available to test a predicted effecton splicing

c.814 + 153_156delGTTT 14/70 Not tested No No—deeply intronic and acommon SNP

c.814 + 237 C > T 6/70 Not tested rs3731424 No—deeply intronic and acommon SNP

c.923 + 62G > A 1/70 Not tested No Not likely—deeply intronicc.1233 + 35 G > A 34/70 Not tested rs521102 No—deeply intronic and a

common SNP

a Genbank Accession No. NM_001274b Variants have been previously reported in SNPper (http://snpper.chip.org/bio/)

Table 4 PPP2R1B variants identified in non-BRCA1/2 cases and sporadic tumours

Nucleotidechangea

Proteineffect

No. of heterozygousfamilial breast cancercases

No. in sporadicbreast tumours

Reported inSNPper

Likely pathogenicity

c.114 + 22C > G

1/72 0/43 No Not likely—deeply intronic

c.115-95 C > A 0/72 2/43 No No—deeply intronic and found in thegermline of the sporadic cancer case

c.206-26 A > G 33/72 15/43 rs7106104 No—intronic and common SNPc.216 T > C p.Y72Y 3/72 1/43 No No—silent SNP, found in germline of the

sporadic cancer case and no predictedeffect on splicing

c.269 G > A p.G90D 0/72 1/43 rs1805076 No—reported SNP and no LCL availableto test the predicted effect on splicingbut found in the germline of the sporadiccancer case

c.539 + 44T > C

3/72 1/43 No No—deeply intronic and common

c.1162-32 A > T 33/72b 15/43b rs4936675 No—common SNP and deeply intronicc.1555-38 A > T 0/72 1/43 No No—deeply intronic and found in the

germline of the sporadic cancer casec.1790-6 C > T 0/72 1/43 No No—no predicted changes in splicing

and found in the germline of thesporadic cancer case

c.1825 G > C p.V609L 10/72 1/43 No No—common SNP and found in thegermline of the sporadic cancer case

c.1867 A > T p.N623S 0/72 2/43 No No—found in the germline of the sporadiccancer cases

*20delGA 3/72 1/43 No No—relatively common 3¢UTR variant

a GenBank Accession No. NT_033899b Homozygotes well differentiated on DHPLC and confirmed by sequencing. Counts shown include eight and two PPP2R1B c.1162 –32 homozygote T cases, respectively

No healthy controls were genotyped for these variants because other evidence indicated that none were somatic mutations orsusceptibility variants


123

(Table 4). In silico analysis of the two unreported

exonic variants and the variant in the 3¢UTR indi-

cated that none were likely to have functional con-

sequences and the other four variants were deeply

intronic. We also identified five additional PPP2R1B

variants among 43 sporadic breast tumours, all of

which we subsequently showed were present in the

matching germline DNA samples. No LCL was

available from the individual with a sporadic tumour

which carried c.269 G > A to test the predicted loss

of two donor consensus splice sites. This variant has

previously reported as being associated with heredi-

tary breast cancer [26].

We identified 10 different variants in PPP2R5B

(Table 5) in index cases from non-BRCA1/BRCA2

families but none of these were in coding regions or

predicted to have functional significance. We also

identified five additional variants, including two in ex-

ons, in the sporadic breast tumours. We found that

c.165 C > T was a silent germline variant (N55N), but

we could not determine the origin of the c.1280 C > G

(T427S) variant because no matching germline DNA

was available.

We identified seven variants in the EIF3S6/Int-6

gene in index cases of non-BRCA1/BRCA2 families.

These included two potentially pathogenic variants but

Table 5 PPP2R5B variants identified in non-BRCA1/2 cases and sporadic tumours

Nucleotidechangea

Proteineffect

No. of heterozygousfamilial breastcancer cases

No. in sporadicbreast tumours


c.1-246 A > T 2/72 3/43 Not tested No No—relatively common SNPand deeply intronic

c.1-11 G > T 0/72 1/43 Not tested No No—no predicted changes in splicing,and found in the germline of thesporadic cancer case

c.165 C > T p.N55N 0/72 1/43 Not tested No No—silent SNP, found in thegermline of the sporadiccancer case and no predictedeffect on splicing

c.200-43 G > A 0/72 1/43 Not tested No Not likely—deeply intronic,but no germline DNAavailable for testing thesporadic cancer case

c.396 + 94 G > A 1/72 0/43 Not tested No Not likely—deeply intronicc.397-13 C > T 1/72 0/43 Not tested No Not likely—intronic and

no predicted changesin splicing

c.592-36delT 1/72 0/43 Not tested No No—deeply intronicc.783-24 T > C 1/72 0/43 Not tested No No—deeply intronicc.783-3 C > T 2/72 4/43 Not tested No No—relatively common variantc.1244 + 19 C > T 0/72 1/43 Not tested No No—no predicted changes

in splicing and foundin the germline of thesporadic cancer case

c.1280 C > G p.T427S 0/72 1/43 0/170 No ?—No germline DNA available.c.1346 +

49 G > A22/72c 16/43 66/170 rs675090 No—common SNP and

deeply intronic*279 C > T 3/72d 2/43 1/161 No No—3¢UTR and relatively

common SNP and foundin the germline of thesporadic cancer caseand in controls

*357 C > T 1/72 1/43 2/161 No No—3¢UTR and presentin controls. No germlineDNA available

*559 G > C 1/72 0/43 0/161 No Not likely—3¢UTR

a GenBank Accession No. NT_033903b Variants have been previously reported in the SNP database SNPper (http://snpper.chip.org/bio/)c Includes one case c.1346 + 49 G > A homozygoted Includes two cases *297 C > T homozygotes


123

further analysis in controls showed that they were un-

likely to be pathogenic (Table 6).

Discussion

Two recent studies of ATR in non-BRCA1/2 families

failed to identify any clearly pathogenic mutations [41,

42]. However, the value of the Finnish study was lim-

ited by the fact that DNA from additional family

members was not available for genotyping. In our

analysis of the ATR gene, we only identified three non-

conservative missense variants and one predicted

splicing variant as potentially pathogenic. However,

genotyping of additional family members and controls

indicated that the c.2437 A > G (p.M813V) and c.2875

G > A (p.V959M) variants are not the underlying

cause of breast cancer in these families, as none of the

other affected individuals carried the variant (c. 2437

A > G: p.M813V; odds against causality of 6.8:1) or the

variant was found in controls (c.2875 G > A:

p.V959M). The overall odds of causality for c.4846

T > G (p.S1616A) variant was 4.3:1 in the two families

in which it segregates, which gives only weak support

for pathogenicity. Genotyping of the two families car-

rying the c.7041 + 8 G > A variant provided no evi-

dence that it is a high-risk variant (overall odds against

causality of 1.5:1).

We identified four rare CHK1 variants among 69

index cases screened, 11 of which came from families in

which the affected family members shared a haplotype

around CHK1. The rare, novel CHK1 variants

(c.354G > A, c.424 + 74T > C, c.718 + 10delT and

c.923 + 62G > A) were not detected in the haplotype-

sharing families. Further analyses suggested that none

of these four variants were likely to be pathogenic. A

number of novel variants were identified in PPP2R1B

and PPP2R5B in the 72 non-BRCA1, non-BRCA2

breast cancer families, and in the sporadic tumour

samples but again further analyses showed that these

were unlikely to be pathogenic.

Mutation analysis of the EIF3S6/Int-6 gene identi-

fied four novel variants in the 67 non-BRCA1/BRCA2

Australian breast cancer families, two of which were

deeply intronic and so not investigated further. The

predicted splicing variant, c.952-5 T > C, found in two

index cases and in 2/175 controls, was assumed to be a

rare, neutral SNP. The *135delT variant was also

identified in two index cases, and is located within the

3¢UTR. Since the 3¢UTR has been implicated in

mRNA signalling and stability, translational control

and binding of microRNAs [43, 44], the *135delT

variant was further examined in 175 normal controls,

where it was found in two samples. This suggests that

the *135delT variant is also a rare, non-pathogenic

polymorphism.

We were also interested in the possibility that vari-

ants in CHK1 (at 11q23–q25), PPP2R1B (11q22) or

PPP2R5B (11q12–q13) might co-segregate in certain

families with ATM (11q22.3) variants. We have pre-

viously identified two multiple-case breast cancer

families [45] carrying ATM c.1066-6T > G and analysis

of these two families suggested that the variant con-

ferred significant increase in risk with penetrance to

age 70 of 78% (95% CI: 36–99%) [33]. Subsequently,

we identified three additional families with ATM

Table 6 EIF3S6 variants identified in non-BRCA1/2 cases

Nucleotide changea Protein effect No. of heterozygousfamilial breast cancercases


c.205 + 25 A > G 30/67 Not tested rs667742 No—common SNPand intronic

c.472-61 A > C 1/67 Not tested No Not likely—deeplyintronic

c.871 + 33 del T 29/67 Not tested No No—common SNPand deeply intronic

c.952-5 T > C Predicted splicingdefect

2/67 2/175 No No—found in controls

c.1165-22 A > G 29/67 85/175 rs630492 No—common SNPand intronic

c.*135delT 2/67 2/175 No No—in 3¢UTR andfound in controls

c.*221 T > G 14/67 99/175 rs603571 No—common SNP

a Genbank Accession No. NT_008046b Variants have been previously reported in the SNP database SNPper (http://snpper.chip.org/bio/)


123

c.1066-6 T > G. The best-fitting Hazard Ratio for all

five c.1066-6 T > G families was 3.4 (95% CI: 0.80–

11.0, P = 0.1), equivalent to a penetrance of 17.2%

(95% CI: 4.7–37.5%) to age 70 i.e. no significant in-

crease in risk [46]. However, there was substantial

heterogeneity between families (P = 0.009). The best-

fitting Hazard Ratio for the three additional families

reported by Thompson et al. was just 0.51 (95% CI:

0.03–3.5, P = 0.5), whereas the best-fitting Hazard

Ratio for the two families included in the original study

was 11.0 (P = 0.003) [46]. We therefore hypothesised

that variants in CHK1, PPP2R1B or PPP2R5B might

co-segregate with ATM c.1066-6 T > G within the first

two families we identified, and that this could explain

the observed heterogeneity of risk associated with

ATM c.1066-6 T > G. However, we did not identify

any variants in CHK1, PPP2R1B or PPP2R5B in these

two families and therefore found no evidence to sup-

port this hypothesis. There is now considerable evi-

dence that ATM c.1066-6 T > G is a neutral variant

[47], and so the segregation between this variant and

breast cancer in these two families is likely to be either

due to chance, or because of mutations in another gene

nearby.

Given that DHPLC is a robust and sensitive

screening technique, it is unlikely that we missed any

coding or splice-site pathogenic mutations among the

samples analysed. In particular, we analysed each PCR

fragment at all the temperatures recommended by the

DHPLC melt algorithm and at 2�C above for EIF3S6/

Int-6, and under these conditions DHPLC has been

reported to have a sensitivity of greater than 99% [35–

37]. It appears unlikely, therefore, that ATR, CHK1,

PPP2R1B, PPP2R5B or EIF2S6/Int-6 account for

more than a small proportion of inherited forms of

breast cancer, if any. However, many rare, novel SNPs

were identified in these genes, and large association

studies of breast/ovarian cancer cases and controls is

warranted to determine whether any of these variants

confer small risks of breast and ovarian cancer.

Acknowledgements We wish to thank David Goldgar forperforming the Bayes analysis, Heather Thorne, Eveline Nie-dermayr, all the kConFab research nurses and staff, and theClinical Follow Up Study (funded by NHMRC Grants 145684and 288704) for their contributions to this resource, and themany families who contribute to kConFab. kConFab is sup-ported by grants from the National Breast Cancer Foundation,the National Health and Medical Research Council (NHMRC)and by the Queensland Cancer Fund, the Cancer Councils ofNew South Wales, Victoria, Tasmania and South Australia andthe Cancer Foundation of Western Australia. We would like tothank all the Australian Red Cross Blood Services (ARCBS)donors who participated as healthy controls in this study, Ra-chelle Morris and the staff at the ARCBS for their assistancewith the collection of risk factor information and blood samples,

Helene Holland for data management, and Joanne Young,Melanie Higgins, Kimberly Hinze, Robert Smith, Judith Cle-ments, Melissa Barker, Rebecca Magson, Genevieve Birney andall the other members of the Molecular Cancer EpidemiologyLaboratory, for their assistance with collection and processing ofblood samples. Sporadic tumour specimens were provided by thePeter MacCallum Cancer Centre Tissue Bank, a member of theABN-Oncology group, which is supported by National Healthand Medical Research Council funding. This work is supportedby a program grant from the NHMRC. ABS is funded by anNHMRC Career Development Award and KKK and GCT areNHMRC Principal Research Fellows.

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mutation analysis of five candidate genes in familial breast cancer

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