Open Access

RAD51, XRCC3, and XRCC2 mutation screening in Finnish breast cancer families

  • Liisa M Pelttari1,
  • Johanna I Kiiski1,
  • Salla Ranta1,
  • Sara Vilske1,
  • Carl Blomqvist2,
  • Kristiina Aittomäki3 and
  • Heli Nevanlinna1Email author
SpringerPlus20154:92

https://doi.org/10.1186/s40064-015-0880-3

Received: 6 February 2015

Accepted: 6 February 2015

Published: 24 February 2015

Abstract

Majority of the known breast cancer susceptibility genes have a role in DNA repair and the most important high-risk genes BRCA1 and BRCA2 are specifically involved in the homologous recombination repair (HRR) of DNA double-strand breaks. A central player in HRR is RAD51 that binds DNA at the damage site. The RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 facilitate the binding of RAD51 to DNA. While germline mutations in RAD51C and RAD51D are associated with high ovarian cancer risk and RAD51B polymorphisms with breast cancer, the contribution of RAD51, XRCC3, and XRCC2 is more unclear. To investigate the role of RAD51, XRCC3, and XRCC2 in breast cancer predisposition and to identify putative recurrent founder mutations in the Finnish population where such mutations have been observed in most of the currently known susceptibility genes, we screened 182 familial Finnish breast or ovarian cancer patients for germline variation in the RAD51and XRCC3 genes and 342 patients for variation in XRCC2, with a subset of the patients selected on the basis of decreased RAD51 protein expression on tumors. We also performed haplotype analyses for 1516 breast cancer cases and 1234 controls to assess the common variation in these genes. No pathogenic mutations were detected in any of the genes and the distribution of haplotypes was similar between cases and controls. Our results suggest that RAD51, XRCC3, and XRCC2 do not substantially contribute to breast cancer predisposition in the Finnish population.

Keywords

Breast cancer RAD51 XRCC3 XRCC2

Introduction

Most of the known breast cancer susceptibility genes function in DNA damage repair. The most important predisposition genes BRCA1 and BRCA2, conferring high life-time risks of breast and ovarian cancer, are involved in the homologous recombination repair (HRR) of DNA double-strand breaks (DSB) (Mavaddat et al. 2010). The moderate-penetrance genes ATM, CHEK2, PALB2, and BRIP1 also have a role in DNA repair. A large proportion of the unexplained familial risk of breast cancer is likely explained by clustering of several common low-penetrance variants and so far, large number of low-risk loci have been identified (Michailidou et al. 2013). However, the currently known high, moderate, and low-penetrance alleles together only explain approximately 35% of the familial risk of breast cancer and thus, other susceptibility loci are likely to exist and genes involved in the homologous recombination repair are attractive candidates.

A central player in the homologous recombination is the RAD51 recombinase that binds to single-stranded DNA at break sites (Suwaki et al. 2011). The binding of RAD51 to DNA is facilitated by several proteins including BRCA2 and the five RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. Deleterious germline mutations in the RAD51C and RAD51D genes confer an increased risk of ovarian cancer (Loveday et al. 2011, 2012) whereas common polymorphisms in the RAD51B gene are associated with male and female breast cancer (Figueroa et al. 2011; Orr et al. 2012). The contribution of RAD51, XRCC3, and XRCC2 to breast cancer susceptibility remains unclear. Deleterious germline mutations in the XRCC2 gene have been identified in exome sequencing of familial breast cancer patients but the association was not confirmed in a larger case–control study (Park et al. 2012; Hilbers et al. 2012). Several association studies of XRCC3 have yielded controversial results yet a meta-analysis by He et al. suggests an association between common XRCC3 polymorphisms and breast cancer risk (He et al. 2012). A likely deleterious missense mutation in the XRCC3 gene has been identified in one breast and ovarian cancer family (Golmard et al. 2013). In the RAD51 gene, one possibly disease-associated missense mutation has been identified in bilateral breast cancer patients whereas three studies report no deleterious RAD51 mutations among breast cancer cases (Kato et al. 2000; Lose et al. 2006; Rapakko et al. 2006; Le Calvez-Kelm et al. 2012).

The presence of recurrent founder mutations in the Finnish population creates an advantage for the identification of new susceptibility genes. We have previously identified Finnish founder mutations in the ovarian cancer susceptibility genes RAD51C and RAD51D (Pelttari et al. 2011, 2012) and recently, we identified a recurrent nonsense mutation in the FANCM gene that associated especially with triple-negative breast cancer (Kiiski et al. 2014). In Finland, recurrent mutations explain most of the familial breast cancer risk caused by the currently known susceptibility genes, such as BRCA1, BRCA2, PALB2, and CHEK2 (Sarantaus et al. 2000; Erkko et al. 2007; Vahteristo et al. 2002), whereas in other more diverse populations several rare mutations in each gene have been identified.

Inactivating mutations in tumor suppressor genes usually lead to decreased protein expression and further to tumor progression (Vogelstein and Kinzler 2004). Loss-of-function mutations have been identified in all the known breast cancer susceptibility genes involved in DNA damage response. We have previously shown that carriers of the truncating CHEK2 c.1100delC mutation often have reduced or absent CHEK2 protein expression in breast tumors (Vahteristo et al. 2002). We have also previously identified two germline mutations in the MRE11 gene among breast cancer patients whose tumors showed decreased expression of the MRN complex proteins MRE11, RAD50, and NBS1 that play an important role in the DNA damage response (Bartkova et al. 2008). The breast tumors were studied by immunohistochemical staining of MRE11, RAD50, and NBS1, and patients whose tumors had reduced expression of all three proteins were selected for further germline DNA analysis. These results indicate that loss or reduction of protein expression in the tumor may be a sign of underlying inactivating germline mutations.

To evaluate the contribution of RAD51, XRCC3, and XRCC2 mutations to breast cancer predisposition, we screened 182 familial Finnish breast or ovarian cancer patients for germline variation in the RAD51 and XRCC3 genes and 342 patients for the XRCC2 gene. To facilitate the mutation discovery, a subset of the patients was selected on the basis of decreased RAD51 protein expression on their breast tumors. We also studied the common variation in these genes with a haplotype analysis in 1516 breast cancer cases and 1234 controls.

Materials and methods

Subjects

The patient samples originated from two unselected series of breast cancer cases and additional familial breast and ovarian cancer patients collected at Helsinki University Hospital Departments of Oncology and Clinical Genetics (Eerola et al. 2000; Fagerholm et al. 2008). The unselected breast cancer cases were ascertained at Helsinki University Hospital Department of Oncology in 1997–1998 and 2000 (n = 884) (Syrjäkoski et al. 2000; Kilpivaara et al. 2005) and Department of Surgery in 2001–2004 (n = 986) (Fagerholm et al. 2008) including 79% and 87%, respectively, of all consecutive, newly diagnosed breast cancer cases during the collection periods. BRCA1 and BRCA2 mutation carriers were excluded from the familial patient series as previously described (Vahteristo et al. 2001, 2002; Vehmanen et al. 1997). RAD51 protein expression was analyzed in 1240 paraffin-embedded invasive breast tumors from these patients as described (Fagerholm et al. 2013).

The RAD51 and XRCC3 genes were screened in 182 and the XRCC2 gene in 342 BRCA1/2-negative familial breast or ovarian cancer patients. Out of these, 71 were selected on the basis of absent or decreased RAD51 expression on tumors. The RAD51-XRCC3 screening included two ovarian cancer probands and four cases affected with both breast and ovarian cancer and the XRCC2 screening included five cases with breast and ovarian cancer; the rest of the screened patients were breast cancer cases. The patients had a strong family background of breast cancer with at least three breast or ovarian cancers among first or second degree relatives, including the proband. A haplotype analysis was performed in 1516 breast cancer cases (including 592 familial BRCA1/2-negative patients) and 1234 population controls that had been genotyped on the iCOGS chip (Michailidou et al. 2013). The population controls were healthy female blood donors from the same geographic region.

This study was performed with written informed consents from the patients and with permission from the Ethical review board of Helsinki University Hospital.

Sequencing

The protein coding regions of the RAD51, XRCC3, and XRCC2 genes were amplified by PCR in genomic DNA samples isolated from peripheral blood of the patients. The primers were designed with Primer3 software (http://bioinfo.ut.ee/primer3/). The PCR conditions are described in Additional file 1: Table S1. The PCR fragments were purified with ExoSAP-IT (Affymetrix) and subsequently sequenced using ABI BigDyeTerminator 3.1 Cycle Sequencing kit (Life Technologies). The capillary sequencing was performed at the Institute for Molecular Medicine Finland (FIMM), University of Helsinki, using 3730xl DNA Analyzer (Life Technologies). The sequence chromatograms were analyzed with FinchTV (Geospiza) and Variant Reporter software (Life Technologies).

Bioinformatics and statistical methods

The pathogenicity of identified missense variants was evaluated with MutationTaster (Schwarz et al. 2010), SIFT, and PON-P (Olatubosun et al. 2012). The haplotype analysis was performed using PHASE v2.1.1 software (Stephens et al. 2001; Stephens and Scheet 2005) and the frequencies of haplotypes were compared between all breast cancer cases versus controls and familial breast cancer cases versus controls. The haplotypes were constructed using all single-nucleotide polymorphisms (SNPs) included in the iCOGS chip (Michailidou et al. 2013) that were located at the RAD51 (n = 14), XRCC3 (n = 10), and XRCC2 (n = 10) gene loci and were not monomorphic in our study population. To test the association of the individual polymorphisms included in the haplotype analysis with breast cancer risk, two-sided p-values with odds ratios (OR) and 95% confidence intervals (CI) for each SNP were calculated using χ2 test or Fisher’s exact test when the count in any of the cells was five or less. Bonferroni’s adjustment was used for multiple-testing correction. We also studied the association of the missense mutations with 10-year breast cancer-specific survival using univariate Cox’s proportional regression models. The follow-up times were left-truncated at the date of ascertainment to account for the latency between diagnosis and study recruitment. The association analyses were performed using the R version 3.0.2 statistical software (http://www.r-project.org/).

Results

In the sequencing of the RAD51 gene, only intronic and untranslated region (UTR) variants were identified. In XRCC3 and XRCC2, one known missense variant was identified in each gene (Table 1). Both missenses were predicted to be polymorphisms, tolerated, and neutral by MutationTaster, SIFT, and PON-P, respectively, and both were detected at comparable frequencies (31.3% for rs861539 in XRCC3 and 4.7% for rs3218536 in XRCC2) as in the Finnish population of the 1000Genomes (31.7% for rs861539 and 4.8% for rs3218536) and of the Exome Aggregation Consortium (ExAC) (31.8% for rs861539 and 3.5% for rs3218536) (Exome Aggregation Consortium (ExAC), Cambridge, MA; http://exac.broadinstitute.org [January 2015]), and in the Sequencing Intiative Suomi (SISu) (30.1% for rs861539 and 3.9% for rs3218536) (http://sisu.fimm.fi/ [January 2015]) (Lim et al. 2014) dataset.
Table 1

Identified germline variants in RAD51, XRCC3, and XRCC2 genes

Gene

Genomic location a

HGVS b

Function

rs-number

AA c

Aa d

Aa e

MAF f

1000G-FIN MAF g

RAD51

15:40987528

c.-98G > C

5´UTR

rs1801320

154

27

1

0.080

0.113

RAD51

15:40987565

c.-61G > T

5´UTR

rs1801321

103

56

23

0.280

0.312

RAD51

15:40987568

c.-58C > G

5´UTR

 

181

1

0

0.003

 

RAD51

15:40987725

c.-3 + 102C > T

intronic

rs3092981

151

22

9

0.110

0.183

RAD51

15:40991153

c.87 + 110A > G

intronic

rs2304579

153

28

1

0.082

0.113

RAD51

15:40998303

c.226-72delA

intronic

rs55943660

156

26

0

0.071

0.108

RAD51

15:40998342

c.226-33 T > G

intronic

rs45457497

136

43

3

0.135

0.129

RAD51

15:41001187

c.344-36 T > G

intronic

rs45455000

153

26

3

0.088

0.108

RAD51

15:41020898

c.531-12C > T

intronic

 

181

1

0

0.003

 

XRCC3

14:104177282

c.55 + 88C > G

intronic

 

181

1

0

0.003

 

XRCC3

14:104174944

c.108G > A

p.(=)

 

181

1

0

0.003

 

XRCC3

14:104174824

c.193 + 34C > T

intronic

rs1799795

171

11

0

0.030

0.032

XRCC3

14:104173300

c.406 + 40C > T

intronic

rs374684710

177

5

0

0.014

 

XRCC3

14:104169435

c.561 + 75G > A

intronic

 

181

1

0

0.003

 

XRCC3

14:104165753

c.722C > T

p.(Thr241Met)

rs861539

91

68

23

0.313

0.317

XRCC3

14:104165647

c.774 + 54G > A

intronic

rs150986165

181

1

0

0.003

0.005

XRCC3

14:104165611

c.774 + 90G > T

intronic

 

181

1

0

0.003

 

XRCC3

14:104165465

c.821 + 5G > A

intronic

 

181

1

0

0.003

 

XRCC3

14:104165411

c.822-57C > T

intronic

rs17101777

181

1

0

0.003

 

XRCC3

14:104165107

c.*28C > T

3'UTR

 

181

1

0

0.003

 

XRCC3

14:104165100

c.*35A > G

3'UTR

 

181

1

0

0.003

 

XRCC2

7:152373252

c.-88G > C

downstream

rs3218384

203

117

22

0.235

0.204

XRCC2

7:152373233

c.-69 T > G

5'UTR

rs3218385

324

16

2

0.029

0.032

XRCC2

7:152357877

c.40-10C > T

intronic

rs3218472

333

9

0

0.013

0.011

XRCC2

7:152346007

c.563G > A

p.(Arg188His)

rs3218536

310

32

0

0.047

0.048

aThe genomic location is denoted according to NCBI37/Hg19 genome build and the variant coding refers to transcripts ENST00000267868 in RAD51, ENST00000352127 in XRCC3, and ENST00000359321 in XRCC2; bvariant description according to HGVS nomenclature; number of ccommon homozygotes, dheterozygotes, and erare homozygotes; fminor allele frequency (MAF) observed in this study; gMAF in 1000Genomes Finnish population.

The association of RAD51, XRCC3, and XRCC2 haplotypes with breast cancer risk was studied among 1516 breast cancer cases (including 592 familial cases) and 1234 population controls. The haplotypes were constructed with PHASE v2.1.1 software using 14 polymorphic sites for RAD51 and ten for XRCC3 and XRCC2. Eleven RAD51, twelve XRCC3, and eight XRCC2 haplotypes were predicted among the samples (Table 2). The distribution of the haplotypes did not differ between all the breast cancer cases and controls (p = 0.45, p = 0.49 and p = 0.55 for RAD51, XRCC3, and XRCC2, respectively) nor between the familial cases and controls (p = 0.66, p = 0.14 and p = 0.80 for RAD51, XRCC3, and XRCC2, respectively). We also tested the association of individual SNPs included in the haplotype analysis with breast cancer but none of them showed significant association (p = 0.060-0.951) (Table 3). After Bonferroni’s correction for multiple testing, p-value < 0.00167 was considered significant.
Table 2

Detected haplotypes with frequency estimates for population controls and breast cancer cases

RAD51 haplotype

Haplotype count

Frequency controls

Frequency all cases

Haplotype count

Frequency controls

Frequency fam cases

GCGCACTTAGAGAC

1510

26.66%

28.11%

999

26.66%

28.80%

GCGCATTTGGAGAC

1510

27.22%

27.63%

996

27.22%

27.36%

GCGTACTTAGGGAC

956

18.07%

16.81%

656

18.07%

17.74%

GTGCACTTAGAGAC

913

16.25%

16.89%

579

16.25%

15.03%

CCGCGCCGAAAGGG

431

8.51%

7.29%

301

8.51%

7.69%

GCTCATTTGGAGAC

138

2.76%

2.32%

97

2.76%

2.45%

GTGCACTTAGATAC

37

0.45%

0.86%

21

0.45%

0.84%

GCGCACTTAGGGAC

2

0.04%

0.03%

2

0.04%

0.08%

CCGCGCTTAGAGAC

1

0.04%

0%

1

0.04%

0%

GCGCACTTGGAGAC

1

0.001%

0.02%

0

0%

0%

CCGCGCCGAAAGAC

1

0%

0.03%

0

0%

0%

 

All BC cases vs controls: p= 0.45

Familial BC cases vs controls: p= 0.66

XRCC3 haplotype

Haplotype count

Frequency controls

Frequency all cases

Haplotype count

Frequency controls

Frequency fam cases

CATGCGCGGG

1599

28.57%

29.58%

1071

28.56%

31.07%

TACGCGCTGG

1586

28.32%

29.25%

1046

28.32%

29.30%

CGTACGCGGG

1159

22.33%

20.05%

778

22.33%

19.17%

CATGCGCGGA

602

10.82%

10.98%

378

10.83%

9.26%

CGTGCGTGGG

227

4.13%

4.03%

151

4.14%

3.98%

TACGCGCTAG

200

3.28%

3.92%

139

3.28%

4.90%

CGTGCGCGGG

75

1.49%

1.25%

55

1.49%

1.52%

CGTACACGGG

29

0.49%

0.56%

18

0.49%

0.51%

CGTGTGCGGG

17

0.41%

0.23%

12

0.41%

0.17%

CGTGCGTGGA

4

0.12%

0.11%

2

0.11%

0.03%

TGCGCGCTGG

1

0.05%

0.005%

1

0.05%

0.003%

CATGCGCTGG

1

0%

0.02%

1

0%

0.04%

 

All BC cases vs controls: p= 0.49

Familial BC cases vs controls: p= 0.14

XRCC2 haplotype

Haplotype count

Frequency controls

Frequency all cases

Haplotype count

Frequency controls

Frequency fam cases

GGGCGCACCT

3633

66.79%

65.48%

2438

66.80%

66.73%

GGGCGCACCG

1253

22.53%

22.97%

814

22.53%

21.79%

GGACGCACCT

247

4.13%

4.78%

159

4.13%

4.81%

GGGCCCATGT

123

2.15%

2.31%

84

2.15%

2.62%

AGGTGGACCT

123

2.25%

2.21%

83

2.25%

2.28%

GAGCGCGCGT

119

2.11%

2.21%

73

2.11%

1.77%

GGGCGCACGT

1

0.02%

0%

1

0.02%

0%

GAGCGCACCT

1

0%

0.02%

0

0%

0%

 

All BC cases vs controls: p= 0.55

Familial BC cases vs controls: p= 0.80

BC = breast cancer; Fam = familial.

Separate analyses were performed for all breast cancer cases versus controls and familial breast cancer cases versus controls. The SNPs included in the analysis are described in Table 3.

Table 3

SNPs from the haplotype analysis with ORs and p -values for breast cancer association

Gene

rs-number

HGVS

MAF controls

MAF cases

OR

95% CI

p- value

RAD51

rs1801320

c.-98G > C

0.09

0.07

0.82

0.66-1.01

0.184

RAD51

rs3092981

c.-3 + 102C > T

0.17

0.18

1.07

0.91-1.27

0.614

RAD51

rs5030791

c.-3 + 203G > T

0.03

0.02

0.83

0.59-1.18

0.583

RAD51

rs2619681

c.-3 + 1398 T > C

0.18

0.17

0.88

0.75-1.05

0.352

RAD51

rs2304579

c.87 + 110A > G

0.09

0.07

0.82

0.67-1.01

0.184

RAD51

rs4924496

c.225 + 1936 T > C

0.29

0.29

1.06

0.90-1.24

0.549

RAD51

rs45503494

c.343 + 494 T > C

0.09

0.07

0.82

0.67-1.02

0.205

RAD51

rs45455000

c.344-36 T > G

0.09

0.07

0.82

0.67-1.02

0.202

RAD51

rs12592524

c.435 + 2149G > A

0.30

0.30

1.07

0.91-1.25

0.518

RAD51

rs4144242

c.436-4016G > A

0.09

0.07

0.82

0.67-1.02

0.202

RAD51

rs4924500

c.530 + 4654A > G

0.18

0.17

0.88

0.74-1.04

0.314

RAD51

rs45532539

c.531-3201G > T

0.004

0.009

1.92

0.97-4.10

0.062

RAD51

rs45507396

c.*929A > G

0.09

0.07

0.82

0.66-1.01

0.187

RAD51

rs45585734

c.*1113C > G

0.09

0.07

0.82

0.66-1.01

0.187

XRCC3

rs861539

c.722C > T

0.32

0.33

1.06

0.90-1.24

0.489

XRCC3

rs861537

c.562-1162G > A

0.29

0.26

0.89

0.76-1.04

0.060

XRCC3

rs861536

c.562-1651 T > C

0.32

0.33

1.06

0.90-1.24

0.475

XRCC3

rs12432907

c.561 + 1132G > A

0.23

0.21

0.92

0.78-1.08

0.092

XRCC3

rs3212092

c.561 + 866C > T

0.004

0.002

0.57

0.20-1.51

0.246

XRCC3

rs3212081

c.407-478G > A

0.005

0.006

1.15

0.55-2.49

0.704

XRCC3

rs3212079

c.407-801C > T

0.04

0.04

0.97

0.73-1.28

0.951

XRCC3

rs861531

c.406 + 533G > T

0.32

0.33

1.06

0.90-1.24

0.459

XRCC3

rs3212042

c.56-652G > A

0.03

0.04

1.17

0.86-1.58

0.456

XRCC3

rs3212028

c.-261 + 1368G > A

0.11

0.11

1.07

0.88-1.29

0.427

XRCC2

rs3218552

c.*1874G > A

0.02

0.02

0.95

0.66-1.37

0.879

XRCC2

rs3218550

c.*1772G > A

0.02

0.02

1.07

0.74-1.55

0.729

XRCC2

rs3218536

c.563G > A

0.04

0.05

1.08

0.82-1.43

0.256

XRCC2

rs3218504

c.122-4868C > T

0.02

0.02

0.94

0.65-1.36

0.878

XRCC2

rs6964582

c.122-5014G > C

0.02

0.02

1.02

0.70-1.47

0.676

XRCC2

rs3218501

c.122-5469C > G

0.02

0.02

0.93

0.64-1.34

0.839

XRCC2

rs3218491

c.121 + 4038A > G

0.02

0.02

1.04

0.72-1.52

0.817

XRCC2

rs3111465

c.40-4608 T > C

0.02

0.02

1.02

0.70-1.48

0.676

XRCC2

rs3094406

c.40-4998G > C

0.04

0.05

0.99

0.76-1.30

0.252

XRCC2

rs3218408

c.39 + 5510 T > G

0.23

0.23

1.09

0.93-1.28

0.447

The SNPs are presented in the same order as in the haplotypes in Table 2.

Since the XRCC2 p.(Arg188His) variant (rs3218536) has been previously associated with poor breast cancer survival (Lin et al. 2011), we performed 10-year breast cancer-specific survival analyses for the XRCC2 p.(Arg188His) missense variant as well as the XRCC3 p.(Thr241Met) (rs861539) variant that were both detected in the sequencing of the genes and also included in the haplotype analysis. Patients with available follow-up information from the sequencing dataset and from the haplotype analysis (n = 1635, events = 106 for XRCC2; n = 1542, events = 80 for XRCC3) were combined for the survival analysis, including 1183 or 1176 cases from the unselected series and 452 or 366 additional familial cases for the XRCC2 and XRCC3 analysis, respectively. Given that most of the familial patients were prevalent cases with more than six months between breast cancer diagnosis and recruitment to the study, the data was left-truncated at the date of ascertainment. Neither of the missenses associated with breast cancer survival (hazard ratio (HR) = 0.67, 95% CI = 0.32-1.40, p = 0.288 for rs3218536; HR = 0.92, 95% CI = 0.66-1.29, p = 0.627 for rs861539).

Discussion

We screened the RAD51, XRCC3, and XRCC2 genes for germline variation in familial BRCA1/2-negative breast or ovarian cancer patients in order to evaluate the role of these genes in breast cancer predisposition in Finland and to identify putative recurrent founder mutations. To facilitate the variant discovery, we selected patients with strong family background of breast cancer from the homogeneous Finnish population where recurrent founder mutations in most of the breast cancer genes are present. In addition, a subset of the patients had decreased RAD51 protein expression on their breast tumors as we hypothesized that loss of protein expression might be a sign of underlying inactivating germline mutations. We also performed haplotype analyses in an extensive series of breast cancer cases and population controls to study the common variation in these genes.

No truncating mutations were identified in any of the genes. In RAD51, only intronic and UTR variants were identified which is in line with the previous studies where no cancer-predisposing mutations were identified among early-onset breast cancer patients (Lose et al. 2006; Rapakko et al. 2006; Le Calvez-Kelm et al. 2012). However, one of the detected 5’UTR polymorphisms, rs1801320, has been found to affect the splicing of RAD51 within the 5’UTR and to modify breast cancer risk among BRCA2 mutation carriers (Levy-Lahad et al. 2001; Antoniou et al. 2007). In XRCC3 and XRCC2, known missense variants p.(Thr241Met) and p.(Arg188His), respectively, were detected. Both of these variants were detected at comparable frequencies as in the Finnish population of the 1000Genomes, ExAC, and SISu datasets and neither was predicted to be pathogenic in silico. A large association study by Breast Cancer Association Consortium (BCAC) found no association with breast cancer risk for neither of the variants (Breast Cancer Association Consortium 2006). However, a meta-analysis by He et al., including also the BCAC study, suggests the XRCC3 p.(Thr241Met) variant is associated with a mild increase in breast cancer risk (OR = 1.10, 95% CI = 1.03-1.16) (He et al. 2012). In our data set, the p.(Thr241Met) and p.(Arg188His) variants did not associate with an increased breast cancer risk nor did they form risk-associated haplotypes. Furthermore, the overall distribution of RAD51, XRCC3, or XRCC2 haplotypes did not differ between all breast cancer cases and controls or between familial cases and controls. Previously, XRCC2 p.(Arg188His) has been associated with poor breast cancer survival (Lin et al. 2011) but in our study, no survival effect was found for this variant or for the XRCC3 p.(Thr241Met) variant.

Like most of the known breast cancer susceptibility genes, RAD51, XRCC3, and XRCC2 also have a role in DNA double-strand break repair by homologous recombination. XRCC2 and XRCC3, two of the five human RAD51 paralogs, help to load RAD51 on the site of DNA damage (Suwaki et al. 2011). XRCC2 gene has been recently linked to breast cancer since rare germline mutations in the gene were identified in breast cancer families (Park et al. 2012). However, no association with breast cancer risk was detected in a subsequent large case–control study (Hilbers et al. 2012) and another study by Golmard et al. (Golmard et al. 2013) reports no pathogenic XRCC2 mutations among early-onset or familial breast cancer patients (Golmard et al. 2013). Interestingly, one Fanconi anemia patient has been found to carry a homozygous truncating XRCC2 mutation (Shamseldin et al. 2012) while biallelic mutations in four breast and ovarian cancer susceptibility genes, BRCA2, BRIP1, PALB2, and RAD51C, are associated with Fanconi anemia (Kee and D'Andrea 2012). Given the unclear role of XRCC2 in breast cancer susceptibility, we sequenced the gene in an extensive series of 342 patients with a strong family history breast cancer. As the only identified coding variant was a neutral missense mutation, our results indicate that XRCC2 is not a major breast cancer susceptibility gene, in line with the studies by Hilbers et al. and Golmard et al. In contrast to XRCC2, no truncating mutations in XRCC3 or RAD51 genes have been reported and only one possibly disease-associated missense in each gene has been detected in breast cancer patients (Golmard et al. 2013; Kato et al. 2000). Furthermore, the RAD51 missense mutation was later also detected once among 1330 breasts cancer cases as well as once among 1123 controls (Le Calvez-Kelm et al. 2012). The absence of mutations in our study as well as the results of the previous studies indicates that XRCC3 and RAD51 are not major breast cancer susceptibility genes.

Conclusions

In conclusion, the absence of mutations among breast cancer families and similar distribution of haplotypes between breast cancer cases and controls suggests that RAD51, XRCC3, and XRCC2 do not substantially contribute to familial breast cancer predisposition in the Finnish population. Taken together, it is unlikely that RAD51, XRCC3, and XRCC2 have a significant contribution to breast cancer susceptibility. However, we cannot exclude possible unique or very rare risk variants.

Declarations

Acknowledgments

We thank research nurses Irja Erkkilä and Virpi Palola for their help with collecting the patient samples and data, Prof. Douglas Easton and Manjeet Bolla for the iCOGS genotyping data, and the Finnish Cancer Registry for the cancer diagnostic data. This study was supported by the Helsinki University Central Hospital Research Fund, the Academy of Finland [132473], the Sigrid Juselius Foundation, the Finnish Cancer Society, Biomedicum Helsinki Foundation, Alfred Kordelin Foundation, and Oskar Öflund Foundation.

Authors’ Affiliations

(1)
Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Biomedicum Helsinki
(2)
Department of Oncology, University of Helsinki and Helsinki University Hospital
(3)
Department of Clinical Genetics, University of Helsinki and Helsinki University Hospital

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