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Human Molecular Genetics, 2003, Vol. 12, No. 8 915-923
DOI: 10.1093/hmg/ddg102
© 2003 Oxford University Press

A naturally occurring mutation in an ATP-binding domain of the recombination repair gene XRCC3 ablates its function without causing cancer susceptibility

Saeed Rafii1, Annika Lindblom2, Malcolm Reed3, Mark Meuth1 and Angela Cox1,*

1Institute for Cancer Studies, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield, S10 2RX, UK, 2 Department Molecular Medicine, Karolinska Institute, S17176 Stockholm, Sweden and 3Academic Unit of Surgical Oncology, Division of Clinical Sciences (South), University of Sheffield Medical School, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, UK

Received January 16, 2003; Accepted February 12, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inherited mutations of the BRCA1 and BRCA2 genes, whose protein products are necessary for the homology-directed DNA repair pathway, confer a dominant susceptibility to cancer. We have investigated whether mutations of genes encoding other components of the same DNA repair pathway can also affect cancer susceptibility. We have identified three novel non-synonymous substitutions in one such gene, encoding the RAD51-related protein XRCC3. One of these variants, D213N, occurs in a highly conserved ATP-binding domain and completely abrogates the ability of the transfected gene to correct the phenotype of XRCC3 deficient cells. The D213N variant was found in the heterozygous state in DNA from 3/1577 healthy individuals. However, we did not detect this variant at all amongst 187 breast cancer families and 1300 unrelated patients with common cancers. Thus we have no evidence that D213N increases the risk of cancer. We propose that not all components of the homologous recombination repair complex can act as cancer susceptibility genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA double-strand breaks (DSB) are one of the most severe forms of DNA damage, leading to chromosome breakage and rearrangement events that can potentially cause tumorigenesis. Cells respond to DSB by inducing cell cycle arrest followed by DNA repair and/or cell death by apoptosis. These responses are crucial for the maintenance of genome stability. Inherited defects of several genes governing the cellular response to DSB confer an increased susceptibility to cancer. These genes include ATM, CHEK2, NBS1 and MRE11, whose proteins are involved in the initiation of the cell cycle checkpoints and DNA repair processes (1). The breast cancer genes BRCA1 and BRCA2 are also required for homology-directed recombination repair of DSB (2,3).

Homologous recombination repair (HRR) is one of two main pathways that repair DNA DSB in mammalian cells (4). In HRR, information is copied from an undamaged homologous template, and thus this pathway is used preferentially during and after S phase, when the DNA has replicated and an undamaged sister chromatid is available (5). Non-homologous end joining (NHEJ), the alternative repair pathway, involves ligation of the exposed DNA ends and does not require any template, but is more error-prone (6). Defects in HRR cause increased chromosomal aberrations such as breaks, gaps and translocations, perhaps by shifting the balance of DSB repair towards NHEJ (7). Much of our knowledge of HRR in eukaryotes comes from studies of S. cerevisiae. The central protein of HRR is Rad51, which is homologous to the E. coli RecA protein (8). In yeast, the Rad55 and Rad57 proteins, that have sequence similarity with Rad51, form a heterodimer that stimulates the efficiency of Rad51-catalysed DNA strand exchange (9). In mammals there are five proteins with homology to RAD51 that are required for HRR; RAD51L1, RAD51L2, RAD51L3, XRCC2 and XRCC3 (10). The homology between the five paralogues is largely confined to the two putative ATP-binding motifs known as the Walker Boxes A and B (11). However the role of ATP-binding in HRR is not clear, since in some cases loss of ATP-binding does not greatly affect HRR (12).

The function of the RAD51 paralogues remains an area of active investigation, but it appears that they are not redundant, but rather co-operate with RAD51 in HRR. Loss of activity of any of the five paralogues is sufficient to impair HRR (13). A number of multi-protein complexes between the various paralogues have been identified. XRCC3, for example, interacts with both RAD51 and RAD51L2 (14–17). Cells lacking functional XRCC3, such as the hamster cell line irs1SF, exhibit decreased HRR resulting in a high rate of spontaneous and induced chromosomal aberrations (18–20). They are also sensitive to ionizing radiation and highly sensitive to DNA cross-linking agents such as cisplatin and mitomycin-C (15,19). These phenotypes are as a result of failure to initiate HRR and aberrant processing of HRR intermediates (21).

Aside from their role in HRR, both the XRCC3 and XRCC2 genes have been proposed as potential tumour suppressors due to their role in chromosome segregation (22). Polymorphisms of these genes that may have a weak effect on protein function have been associated with breast cancer (23,24). In this study, we report three novel rare nucleotide substitutions of XRCC3 present in the South Yorkshire population. We investigated whether these variants, found at conserved sites in and around the Walker B ATP-binding domain, had any effect on XRCC3 activity or cancer susceptibility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Three novel variants in exon 7 of XRCC3
As part of routine genotyping of the previously identified XRCC3 T241M polymorphism (25) in breast cancer cases and controls, we screened exon 7 of the XRCC3 gene in DNA from 1416 women by PCR-SSCP (single-strand conformation polymorphism). This analysis also resulted in the identification of three novel SSCP variants in this exon (Fig. 1). DNA sequence analysis of these revealed three non-synonymous substitutions: C/T (P199L), G/A (D213N ) and G/T (T241I ) (at positions 17941, 17982 and 18068 respectively in GenBank sequence AF037222). The T241I G/T substitution is immediately adjacent to the previously described T241M C/T polymorphism (25). All three novel variants were heterozygous in the individuals that carried them.



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  		Figure 1. Novel variants in exon 7 of XRCC3. Results of SSCP analysis showing the three novel variants compared to the wild-type SSCP pattern in each case.

 
The D213N substitution occurs in a highly conserved domain
The D213N variant occurs in the highly conserved ATP binding domain, known as the Walker box B (11), and the aspartate at this site is conserved between human and mouse XRCC3, human XRCC2 and RAD51 (Fig. 2). Furthermore, alignment of XRCC3 with 23 other orthologues and paralogues of the Rad51 family revealed that this residue is conserved in all family members and species for which nucleotide sequence data is available (data not shown). The XRCC3 P199L and T241M/I variants fall close to this region and these sites are also conserved between human and mouse XRCC3. The alignment data suggest that these residues, and the D213 amino acid site in particular, may be important for the function of XRCC3.



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  		Figure 2. Amino acid sequence alignment. Amino acid sequences of the relevant regions of human RAD51, human XRCC3, mouse XRCC3 and human XRCC2 are shown. Black boxes indicate amino acid identity and grey boxes indicate conservation of charge. The positions of the Walker boxes A and B are shown by a horizontal line and the positions of the amino acid variants investigated are indicated by vertical arrows.

 
The XRCC3 D213N variant is non-functional
Given the disruptive nature of the D213N mutation, in a highly conserved region of XRCC3, we determined whether XRCC3 alleles carrying the D213N substitution were able to correct the sensitivity of XRCC3-deficient cells to agents inducing DSB. We first cloned the human XRCC3 cDNA into the pcDNA 3.1 expression vector under the control of the human cytomegalovirus promoter. We then generated the three novel variant alleles, and the T241M polymorphic allele, by site-directed mutagenesis. The presence of the desired substitution and the absence of any spurious PCR-generated mutations were verified by sequencing each full-length cDNA. The expression constructs for the wild-type and variant XRCC3 alleles were then transfected into irs1SF cells by electroporation. Clones that had incorporated the transfected DNA were identified by virtue of their resistance to the antibiotic G418. Two or four independent clones from each transfection experiment were selected for further study. To verify expression of the introduced human genes in hamster cells, polyA+ mRNA samples extracted from independent transfected clones and the parental irs1SF cells were reverse-transcribed and PCR amplified with primers specific for human XRCC3. For each reverse-transcription experiment, a negative control without reverse transcriptase was also included, to allow detection of potential PCR products arising from any contaminating DNA that might be present. PolyA+ mRNA from the human cell line LM 217E, which expresses functional XRCC3, provided a positive control. Figure 3A shows that a single RT–PCR product of the expected size (1047 bp) is present in a representative clone from each transfection, and no products are present in the corresponding negative controls. DNA sequencing of each amplified product confirmed the expression of the desired human allele (Fig. 3B).



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  		Figure 3. Transfected clones express variant and wild-type XRCC3. (A) To confirm expression of the transfected wild-type or mutant genes, polyA+RNA was extracted from each transfected irs1SF clone and subjected to RT–PCR. The resulting RT–PCR gel image for one representative clone from each transfection is shown (lanes 2, 4, 6, 8 and 10), together with RT–PCR products from the LM 217E human cell line as a positive control (lane 12) and non-transfected irs1SF as a negative control (lane 14). Negative controls for each RT–PCR reaction, lacking reverse transcriptase, were also included (lanes 3, 5, 7, 9, 11 and 13). Lane 1 contains a DNA size marker. (B) DNA sequence chromatograms derived from sequencing the four mutant RT–PCR products shown in (A). The RT–PCR products were sequenced completely in both directions, to confirm the expression of the correct human allele and the absence of extraneous mutations. The position of the relevant base change is indicated with an arrow.

 
To assess whether the variant alleles retained XRCC3 function, we determined whether the transfected DNA was able to correct the sensitivity of irs1SF cells to the DNA cross-linking agent Mitomycin-C (MMC). Consistent with previous published results, cells expressing the wild-type XRCC3 gene show increased resistance to MMC, but are not fully complemented with respect to survival in MMC relative to the parental cell line AA8 (Fig. 4A). The incomplete correction of the phenotype may be a result of using the human allele to complement the defect in hamster cells (19). Alternatively, our expression constructs may not yield levels of XRCC3 sufficient for restitution of full MMC resistance. Cells expressing T241I did not show any significant difference in cell survival relative to those expressing the wild-type. The T241M and P199L variants led to a slight but significant resistance to MMC in relation to the wild type (P=0.02 and 0.005 at the highest dose, respectively). This is most likely to be due to variations in the level of expression of the transfected alleles or variation in the stability of the encoded proteins. Most strikingly, the D213N variant was unable to correct the sensitivity of irs1SF to MMC. Survival of cells expressing this allele was indistinguishable from that of untransfected irs1SF cells, for all four independent isolates tested. As reported previously, the untransfected irs1SF cells are also sensitive to the topoisomerase I inhibitor camptothecin, and the S-phase synchronizing agent thymidine (26). As with MMC, the D213N variant was unable to correct the sensitivity of irs1SF to either of these agents (Fig. 4B and C).



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  		Figure 4. The effect of mutations in XRCC3 on cell survival following exposure to mitomycin-C, camptothecin, and thymidine, in irs1SF and AA8 cells. (A) Mitomycin-C survival curves for XRCC3-deficient irs1SF cells, irs1SF cells stably transfected with wild-type or mutant (D213N, T241M, T241I, P199L) XRCC3 cDNA, and parental AA8 cells. Data are shown as mean and standard deviation of 12 experiments (three experiments on each of four independent cloned transfectants) for D213N and six experiments (three experiments on each of two independent clones) for all other transfectants. For some points the standard deviation is very low and thus error bars are not visible. Plot symbols: ({oplus}) AA8; ({square}) wild-type XRCC3; ({blacklozenge}) irs1SF; ({blacktriangleup}) D213N; ({circ}) T241M; ({triangleup}) P199L; ({blacksquare}) T241I. (B) Camptothecin survival curves for XRCC3-deficient irs1SF cells, irs1SF cells stably transfected with wild-type or mutant (D213N, T241M, T241I, P199L) XRCC3 cDNA, and parental AA8 cells. Other details and plot symbols are as described for (A). (C) Thymidine survival curves for XRCC3-deficient irs1SF cells, irs1SF cells stably transfected with wild-type or mutant (D213N ) XRCC3 cDNA and parental AA8 cells. Other details and plot symbols are as described for (A). (D) Mitomycin-C survival curves for the parental cell line AA8 (containing endogenous functional XRCC3) and AA8 transfected with wild-type or mutant XRCC3 D213N cDNA. Other details and plot symbols are as described for (A).

 
To determine whether D213N exerted a dominant-negative phenotype, we also transfected the Chinese hamster ovary cell line AA8 and the human colorectal carcinoma cell line SW480, both of which contain functional XRCC3. The transfections of wild-type and D213 expression constructs, and verification of expression by sequencing of RT–PCR products, were carried out as described above (data not shown). However, no significant differences in sensitivity to MMC in strains expressing wild type or mutant alleles were detected (results for AA8 are shown in Fig. 4D).

XRCC3 D213N is not associated with sporadic or familial breast cancer
In our initial screen of DNA from 1416 women, only one individual was identified carrying a single novel variant in each case. We developed a 5' nuclease PCR (TaqManTM) assay to allow high-throughput screening for the presence the D213N variant in a panel of 1577 healthy controls, 915 unrelated patients with breast cancer, 131 with colorectal cancer, 98 with bladder cancer, 156 with prostate cancer and 224 familial cancer cases. The latter included cases from 93 high-risk breast cancer families. However, only two further individuals heterozygous for the D213N variant were identified. The D213N substitution generates a novel Hin fI restriction enzyme site. We designed a Hin fI restriction fragment length polymorphism assay and used this to re-confirm the genotypes of all three carriers. None of these individuals showed any evidence of cancer or significant family history of cancer (Table 1). All are from South Yorkshire and we cannot rule out the possibility that they may be distantly related.


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  		Table 1. Population screening for the XRCC3 D213N variant
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have identified three new rare variants of XRCC3, and report their effect on XRCC3 function. One of these, D213N, is in the highly conserved Walker box B, a putative ATP-binding domain. Systematic functional and structural studies of XRCC3 have not yet been carried out. For this reason, the specific functions of the different regions of the XRCC3 protein have not been identified. However, two potential ATP-binding domains, the Walker boxes A and B, have been identified by alignment with the recA homologues. Although the ATPase domain is highly conserved in recA orthologues and paralogues from bacteria to man, the role of ATP hydrolysis in recombination and repair is not well understood. Many amino acid substitutions of residues in the ATP binding site of the E. coli recA protein dramatically reduce the level of homologous recombination and/or recombinational DNA repair (27). Mutations of recA homologues in the yeast S. cerevisiae have varied effects. Mutations of the Walker A/B motifs of RAD51 and RAD55 result in radiation sensitivity as well as meiotic recombination deficiency (28,29) and mutation of the Walker A motif in DMC1 results in a dominant meiotic null mutant (30). In contrast, similar mutations of RAD57 do not display radiation sensitivity and are only modestly defective in meiotic recombination. There have been more limited studies of the effects of mutations of this domain in the human RAD51 paralogues. Mutant alleles of the RAD51 paralogue XRCC2 carrying a site-specific substitution of the Walker box A that eliminated ATP binding activity were able to correct the radiation and MMC sensitive phenotype of hamster XRCC2-deficient cells (12). Similarly chicken DT40 RAD51-deficient cells could be rescued by overexpression of a human RAD51 allele that carried a mutation of the Walker A/B motif that reduced ATP hydrolysis but not ATP binding (31). In the present study, the D213N change replaces the highly conserved acidic aspartate residue with the uncharged polar residue asparagine in the Walker box B, which could potentially disrupt the ATP-binding activity. Our results clearly demonstrate that the D213N substitution has a dramatic effect on XRCC3 function in HRR, and further biochemical studies are required to determine the effect of this substitution on ATP-binding and hydrolysis.

A number of studies have focussed on the potential role of common polymorphisms in HRR genes as low-penetrance cancer susceptibility alleles, but thus far only limited information is available on the functional effects of such alleles (23). The XRCC3 T241M polymorphic variant has been reported to be associated with melanoma, bladder and breast cancer (24,32,33). However, the original melanoma and bladder associations have not been replicated (34,35). This might be explained if XRCC3 T241M itself was not disease causing, but was in linkage disequilibrium (at least in some populations) with a closely linked dysfunctional variant. Two lines of evidence are consistent with this hypothesis. Firstly, a rare haplotype containing the XRCC3 T241M variant was more strongly associated with breast cancer than T241M itself (24). Secondly, the present study and that of Araujo et al. (36) found no detrimental effect of this variant on the ability of a transfected XRCC3 gene to complement the HRR-deficient phenotype of irs1SF cells. Alternatively, the lack of any apparent detrimental effect on function may be due to differences between species, since we have used the human gene to complement the deficiency in hamster cells. We did observe subtle differences in the relative responses of the T241M and T241I variants to MMC and camptothecin (Fig. 4A and B). The T241M variant seemed to confer slightly increased resistance to MMC, whereas the T241I variant yielded the same response as the wild-type. In contrast, the T241M variant conferred increased resistance to camptothecin, but not to MMC. These results require confirmation using a larger number of clones, but if proven, would raise the intriguing possibility that different changes at amino acid position 241 affect the degree of resistance to different DSB-inducing agents.

After screening a large number of individuals and high risk cancer families, we found only three individuals carrying the D213N variant. Two of these were women attending mammography screening, with no evidence of breast lesions, aged 54 and 62. The third was an anonymous male blood donor aged 34 years. The limited family history information available on the three carriers did not yield any evidence that they were from high risk cancer families. Although we cannot exclude the possibility that they may develop cancer later in life, it is clear that these individuals do not fall into the category of either early onset or familial cancer. We would not predict any cellular phenotype in these heterozygous individuals, since the mutant allele does not act in a dominant-negative manner (Fig. 4D). In screening over 1000 breast cancer cases, we have better than 90% power to detect a variant if it were present at the frequency observed in our healthy controls. Thus these data demonstrate that a naturally occurring Walker Box B mutation of XRCC3, which imparts sensitivity to DNA damage, does not confer increased susceptibility to breast cancer, and we have no evidence for any increased risk in the other common cancers screened here either. One possible explanation for this is that loss of RAD51 paralogues may not confer sufficient growth advantage on cells. In this context, the effect of XRCC3 knock-out in mice has not yet been published, but knock-out of XRCC2 and several of the other RAD51 paralogues is embryonic lethal (37). This indicates that cell death rather than proliferation may be the normal outcome of loss of XRCC3 in vivo. Therefore, our data suggest that mutations in genes encoding proteins that are involved in the same repair pathway as BRCA1 and BRCA2, may not confer the same risk of cancer as do mutations in the genes for BRCA1 or 2 themselves.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cancer patients and controls
Genomic DNA was extracted from venous blood samples from subjects recruited as follows. Unrelated breast cancer patients were recruited from surgical outpatient clinics at the Royal Hallamshire Hospital, Sheffield, UK between November 1998 and June 2001. Healthy women attending mammography screening in Sheffield were recruited concurrently. DNA samples from Swedish women with breast cancer and a family history of breast cancer were obtained by A.L. Healthy males and females were recruited from amongst blood donors in Sheffield. Patients with bladder and prostate cancer were recruited from urology clinics at the Royal Hallamshire Hospital Sheffield, and colorectal cancer patients were recruited from those receiving surgery in Sheffield or neighbouring hospitals. Multi-case cancer families were recruited from regional oncology clinics at Weston Park Hospital, Sheffield. Ethical committee approval was obtained from South Sheffield Research Ethics Committee and informed consent was obtained from all subjects.

SSCP screening
DNA amplifications were carried out using 100 ng genomic DNA, 6 pmol of each primer (Table 2) and 18.2 µl 1.1xPCR master mix (AB gene) in a final volume of 20 µl. Cycling conditions were 5 min at 95°C, followed by 35 cycles of 95°C for 35 s, 61°C for 25 s and 72°C for 25 s. A final extension of 5 min at 72°C was also applied. A 1.5 µl aliquot of amplified DNA was mixed with 10 µl SSCP loading dye (95% Formamide, 0.05% EDTA, 0.05% Bromophenol blue and 0.05% Xylene cyanol) and incubated at 95°C for 5 min. Samples were then rapidly cooled on ice, loaded onto a 12% polyacrylamide gel and electrophoresed for 5500 V/h. Gels were stained with 0.1% silver nitrate and developed in 1.5% sodium hydroxide. Sequencing of PCR products containing novel variants was carried out using ABI 377 and Licor 4200IR automated sequencers.


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  		Table 2. Primers and probes
 
TaqManTM genotyping
DNA amplifications for 5' nuclease PCR were carried out using 12.5 µl 2xqPCR mastermix (Eurogentec), 20 ng genomic DNA template, 50 nM of forward and 300 nM of reverse primers, 50 nM FAM-labelled probe and 125 nM TET-labelled probe (Table 2) in a final volume of 25 µl. The cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 63°C for 1 min. Samples were then analysed using the ABI 7200 Sequence Detector to determine levels of FAM and TET fluorescence.

Mammalian cell culture
Wild-type Chinese Hamster Ovary (CHO) XRCC3 proficient (AA8) and XRCC3-deficient (irs1SF) cells were cultured in monolayer in Dulbecco's modified eagle's medium (Life Technologies Inc.) supplemented with 9% fetal calf serum. Cells were grown at 37°C in 5% CO2 humidified incubator. Both cell lines were the kind gifts of Dr Larry Thompson.

Site-directed mutagenesis
Human XRCC3 mRNA (extracted from the LM217E cell line) was reverse-transcribed, amplified using Pfu Turbo polymerase (Stratagene) and cloned into the expression vector pcDNA 3.1/Vs-His Topo TA (Invitrogen). The mutagenic polymerase chain reaction was carried out using QuikChange site-directed mutagenesis kit (Stratagene) with mutagenic primers (Table 2) designed to create the desired changes. All mutations were confirmed by direct sequencing. Approximately 15 µg of plasmids carrying wild-type and each mutant DNA were transfected into irs1SF cells by electroporation (Bio-Rad Gene Pulser at 400 V and 125 µF). A total of 5x105 cells were seeded into 100 mm plates and incubated for 14 days in 100 µg/ml of G418. Two or four independent clones for each construct were picked, expanded and tested in cellular toxicity assays.

Drug toxicity assay
Cell survival assays were performed by exposing individual clones transfected with variant or wild type XRCC3 to the agents MMC, camptothecin and thymidine. Four independent clones (D213N) or two independent clones (other variants) were subjected to the drug toxicity assay. A total of 500 cells for each clone were seeded in triplicate into 100 mm dishes in 10 ml medium containing 100 µg/ml of G418 and incubated at 37°C overnight. Desired concentrations of MMC (0–100 nM), camptothecin (0–75 nM) or thymidine (0–10 mM) were added and plates were incubated for 10–12 days at 37°C. Colonies were stained with Methylene blue and counted. Cell survival curves were obtained by plotting the fraction of plated cells of each clone forming colonies against concentration of the drug, corrected for plating efficiency in the absence of the drug.

RNA extraction and RT–PCR
Messenger RNA was extracted from the same clones for which drug sensitivity had been determined using Oligotex Direct mRNA micro kit (Qiagen) according to the manufacturer instructions. RT–PCR was performed using primers designed from human XRCC3 cDNA (Table 2). These primers were chosen in part to preferentially amplify the human XRCC3 in a hamster background. Sequence analysis of amplified products confirmed that the human XRCC3 transcript was amplified. A negative control (excluding reverse transcriptase) was used for each sample to eliminate the possibility that any PCR products originated from contaminating DNA.

Restriction fragment length polymorphism analysis
Genomic DNA was amplified for XRCC3 exon 7 using the same primers and conditions as for SSCP screening. Restriction digest was carried out by mixing 7 µl of PCR product with 1 µl of bovine serum albumin, 1 µl buffer and 10 units Hin fI (Promega) overlaid under 20 µl mineral oil. The mixture was incubated at 37°C for 2 h. Gel electrophoresis was then carried out by loading 10 µl of the digested PCR product on 1.5% agarose gel.


   ACKNOWLEDGEMENTS
 
We are grateful to Anil Ganesh for invaluable assistance with laboratory techniques, Gordon MacPherson for DNA extraction, and Thomas Helleday for advice and helpful comments on the manuscript. We would also like to thank the individuals who donated blood for this study, Saba Balasubramanian, Helen Cramp, Mark Katory, Dan Fletcher, Jim Catto and Barry Hancock for patient recruitment and sample collection, and Dan Connley for data management. The work was supported by Yorkshire Cancer Research, the Breast Cancer Campaign, and a scholarship from the Iranian Government (S.R.)


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1142712373; Fax: +44 1142713515; Email: a.cox{at}shef.ac.uk Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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