Open Access

Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay

  • Authors:
    • Annelot Baert
    • Julie Depuydt
    • Tom Van Maerken
    • Bruce Poppe
    • Fransiska Malfait
    • Tim Van Damme
    • Sylvia De Nobele
    • Gianpaolo Perletti
    • Kim De Leeneer
    • Kathleen B.M. Claes
    • Anne Vral
  • View Affiliations

  • Published online on: January 25, 2017     https://doi.org/10.3892/or.2017.5407
  • Pages: 1379-1386
  • Copyright: © Baert et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Breast cancer risk drastically increases in individuals with a heterozygous germline BRCA1 or BRCA2 mutation, while it is estimated to equal the population risk for relatives without the familial mutation (non-carriers). The aim of the present study was to use a G2 phase-specific micronucleus assay to investigate whether lymphocytes of healthy BRCA2 mutation carriers are characterized by increased radiosensitivity compared to controls without a family history of breast/ovarian cancer and how this relates to healthy non-carrier relatives. BRCA2 is active in homologous recombination, a DNA damage repair pathway, specifically active in the late S/G2 phase of the cell cycle. We found a significantly increased radiosensitivity in a cohort of healthy BRCA2 mutation carriers compared to individuals without a familial history of breast cancer (P=0.046; Mann-Whitney U test). At the individual level, 50% of healthy BRCA2 mutation carriers showed a radiosensitive phenotype (radiosensitivity score of 1 or 2), whereas 83% of the controls showed no radiosensitivity (P=0.038; one-tailed Fisher's exact test). An odds ratio of 5 (95% CI, 1.07-23.47) indicated an association between the BRCA2 mutation and radiosensitivity in healthy mutation carriers. These results indicate the need for the gentle use of ionizing radiation for either diagnostic or therapeutic use in BRCA2 mutation carriers. We detected no increased radiosensitivity in the non-carrier relatives.

Introduction

BRCA1 and BRCA2 heterozygous mutation carriers have a strongly increased risk to develop breast cancer (BC) and ovarian cancer (OC). The lifetime risk to develop BC is 70–80% for BRCA1 mutation carriers and 50–60% for BRCA2 mutation carriers (1). For relatives who did not inherit the germline BRCA1/2 mutation segregating in the family (non-carrier relatives), the risk of BC occurrence is generally estimated to be as low as the risk assessed in the general population. This may imply that intensified BC detection screening, using, amongst others, mammography screening and MRI, as applied in individuals at high-risk is unnecessary in non-carriers (27). However, one study reported a 2–5-fold increase in BC occurrence in non-carriers of families with either BRCA1 or BRCA2 mutations (8). Another study reported a younger than expected age at diagnosis of BC for non-carriers, that was most evident in BRCA1 families (9). Moreover, one study by Evans et al detected a possible higher relative risk for BC in non-carrier relatives of BRCA2 families, compared to non-carriers in BRCA1 families (10). In summary, these studies suggest that DNA alterations (for example SNPs) in other genes may modify the relative risk for the development of BC in non-carriers, compared to the general population. Moreover, the authors of these studies recommend targeted BC detection screening using for example mammography in non-carriers at a frequency comparable to the intensive BC screening performed in individuals at high-risk.

Both BRCA1 and BRCA2 are caretaker genes playing different roles in the repair of DNA double-strand breaks (DSB), induced by exposure to genotoxic agents such as ionizing radiation (IR). While BRCA1 has a more general function in the detection and signaling of a DSB and in the activation of the G2/M cell cycle checkpoint, BRCA2 exerts a specific function in the recruitment of RAD51 recombinase to the DSB site. This latter event is essential for the activation of the homologous recombination (HR) pathway, that relies on the undamaged sister chromatid as a template for resynthesis of the damaged strand. This occurs in the late S and G2 phase of the cell cycle and leads to error-free repair of DSB (1).

Knowing that both BRCA1 and BRCA2 are important in the repair of DSB, exposure of mutation carriers to IR, a potent inducer of DSB, for either diagnostic or therapeutic purposes appears to be counterintuitive, as mutation carriers may be more prone to develop radiation-induced BC (11).

Radiosensitivity of BRCA1 mutation carriers has previously been reported in the literature and was investigated and confirmed by our research group by means of the G2 micronucleus (MN) assay in combination with an evaluation of the G2/M checkpoint efficiency in peripheral blood lymphocytes of healthy BRCA1 mutation carriers compared to healthy volunteers (12). However, the impact of IR on heterozygous cells of healthy BRCA2 mutation carriers remains to be elucidated.

To date, several cohort studies were able to prove a positive correlation between exposure to diagnostic X-rays and BC risk in BRCA2 mutation carriers (11,13,14). Others however, could not detect a similar correlation (1518). Furthermore, Bernstein et al detected no increased induction of contralateral BC upon exposure to radiotherapy in BRCA2 mutation carriers (19). Such discrepancies are likely due to differences in inclusion criteria, data acquisition and other issues of the studies. It is however difficult and unethical to design long-term unbiased studies to evaluate the relationship between BRCA2 mutations, the exposure to diagnostic or therapeutic radiation and BC risk. In vitro chromosomal assays are effective tools to investigate radiosensitivity. Chromosomal radiosensitivity testing on lymphocytes from BRCA2 mutation carriers has been performed with techniques such as the G0 MN and the G2 assays for chromatid breaks, occasionally enhanced with a whole-chromosome painting FISH (2025). However, for several of these studies, it was unclear whether the BRCA2 heterozygotes were healthy individuals or BC patients, which was previously broached by Baeyens et al (20). Furthermore, differences in the experimental setup make comparisons between studies difficult (26). Despite these differences, all but one study was able to detect an elevated chromosomal radiosensitivity in BC patients with a BRCA2 mutation. However, no comparison was made with sporadic BC patients. The study of Baeyens et al previously demonstrated enhanced radiosensitivity in both BC patients with a BRCA1/2 mutation and sporadic BC patients, suggesting that the enhanced sensitivity may not be the result of the mutation (20). No univocal results were achieved for healthy BRCA2 mutation carriers. Radiosensitivity in non-carrier relatives has not been studied extensively, only one study reported no increased radiosensitivity measured with the G0 MN and G2 chromatid break assay in a small cohort (n=10) of relatives of both BRCA1 and BRCA2 families without the familial mutation when compared to a population cohort (20).

In the present study, we aimed to investigate chromosomal radiosensitivity in healthy BRCA2 mutation carriers by means of the G2 MN assay. We previously used this assay and confirmed radiosensitivity in healthy BRCA1 mutation carriers (n=18) compared to healthy controls without a family history of BC or OC (n=20) (12), and in an ataxia-telangiectasia patient and family members (27). In addition, we also included healthy relatives not carrying the familial germline BRCA1 or BRCA2 mutation in the present study. This cohort of non-carriers was included to evaluate radiosensitivity in individuals with a comparable genetic background, but without the familial BRCA1 or BRCA2 mutation.

Materials and methods

Sample collection

Blood samples were collected from individuals consulting the Centre for Medical Genetics of the Ghent University Hospital (CMG; Ghent, Belgium), in the context of predictive testing for hereditary BC. Heparin blood samples were collected for the G2 MN assay. In addition, EDTA samples were collected for mutation analysis. We collected blood samples from 18 BRCA2 mutation carriers and 17 subjects from both BRCA1 (n=9) and BRCA2 (n=8) families not showing the familial mutation (non-carriers). None of the individuals selected for the present study had developed cancer at the time of the blood sample collection. We also selected 18 blood samples from a historical cohort of healthy volunteers without a personal or familial history of BC or OC for optimal age and gender match, to determine the normal distribution of MN yields in unaffected individuals from the general population (12).

The present study was approved by the Ethics Committee of Ghent University Hospital (B67020111641 d.d. 20/09/2011) and all participants signed an informed consent.

Molecular analysis

All healthy individuals selected for the present study had a family history of BC or OC and a mutation in either BRCA1 or BRCA2 was identified in each proband. All BRCA2 mutation carriers are heterozygous for an unequivocal deleterious mutation. This was confirmed by Sanger sequencing of the relevant amplicon. Sanger sequencing was performed on the ABI3730XL instrument using the BigDye® Terminator Cycle Sequencing kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions; sequences were analyzed using the SeqPilot software (JSI Medical Systems GmbH, Ettenheim, Germany).

Molecular analyses were not performed in healthy volunteers due to the absence of a personal or familial anamnesis for BC or OC.

The G2 MN assay

The G2 MN assay was performed as previously described (12). In brief, heparinized blood was cultured in the presence of phytohaemagglutinin (PHA; 2% v/v; Gibco, Grand Island, NY, USA) to stimulate T-lymphocyte division. After 3 days, a population of cycling lymphocytes was obtained and the culture was irradiated with a 2 Gy dose of 60Co γ-rays. We opted to use a dose of 2 Gy as this is a well-accepted dose for chromosomal radiosensitivity testing in lymphocytes (2022,24). Immediately after irradiation, cytochalasin B (cyto B; 6 µg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to all cultures, including a non-irradiated culture. Cyto B blocks the cytokinesis and allows the identification of first-division cells as a binucleated (BN) cell. After an incubation period of 8 h, all cultures were fixed with the sequential addition of KCl (75 mM), a solution of methanol, acetic acid and Ringer (4:1:5), and a combination of methanol and acetic acid (4:1) to pelleted cells. Finally, the cell suspension was concentrated and spread on slides. Slides were stained with 4′,6-diamidino-2-phenlylindole (DAPI) and scanned with a Metafer 4 platform and MN search software (MetaSystems GmbH, Altlussheim, Germany). The automated image analysis system selects BN cells and determines the number of MN/BN cells. BN cells and MN selection are manually checked for false positives or false negatives. For each condition, 2 cultures were prepared and 2 slides/culture were analyzed. A minimum of 600 BN cells were scored/coded slide. To assess individual radiosensitivity, a radiosensitivity score (RS score) was determined. The mean and SD of the MN yield of the group of healthy volunteers (HV) was set as the cut-off value to determine the RS score of HV, BRCA2 mutation carriers and non-carrier relatives. An MN yield higher than the meanHV + 1SDHV was scored as 1, indicating a milder radiosensitive phenotype, whereas a result higher than the meanHV + 2SDHV was scored as 2, and indicated a more severe radiosensitive phenotype. When the individual value was lower than the meanHV + 1SDHV, a score of 0 was attributed to the tested subject.

Statistical analysis

Age and gender differences among the 3 groups were judged by means of a one-way ANOVA and Chi-square test, respectively. The median, interquartile range, average and standard deviation of micronuclei yields (number of MN/1,000 BN cells) were assessed in each group of subjects. Intergroup differences of MN yields between HV, BRCA2 mutation carriers and non-carrier relatives of BRCA1 and BRCA2 pedigrees were analyzed by the Mann-Whitney-Wilcoxon test. A one-tailed Fishers exact test was performed to compare the unpaired and independent proportion of patients showing a radiosensitive phenotype, evaluated by RS scoring. For both assays a 5% α error was set as the limit for statistical significance. The odds ratio (OR) was calculated, based on the RS scores in healthy individuals and BRCA2 mutation carriers, to assess the association between the presence of a BRCA2 mutation and radiosensitivity according to the following formula:

OR=#BRCA2mutationcarrierswithanRS>0x#healthyvolunteerswithanRS=0#BRCA2mutationcarrierswithanRS=0x#healthyvolunteerswithanRS>0

The 95% confidence interval (CI) was used as a proxy for significance. The VassarStats platform and the SPSS software (IBM, version 23) were used to perform statistical analysis.

Results

The mean age did not significantly differ for the HV (35.3 years), the BRCA2 mutation carriers (40.9 years) and the non-carrier relatives (40.0 years) (P=0.56; one-way ANOVA). In addition, no significant difference in gender distribution was observed for these 3 groups (68, 61 and 71% of the individuals were female, respectively) (P=0.84; Chi-square test). The number of spontaneously occurring micronuclei (MN yields in non-irradiated samples) was not significantly different among the 3 groups of enrolled subjects (Table I and Fig. 1).

Table I.

Overview of the median, interquartile range, mean and SD of the micronucleus yield (#MN/1,000 BN).

Table I.

Overview of the median, interquartile range, mean and SD of the micronucleus yield (#MN/1,000 BN).

Group data0 Gy2 Gy
Healthy volunteers (HV)
  Median1256
  Interquartile range9.7527.5
  Mean14.3361.22
  SD8.8521.73
BRCA2 mutation carriers (MC)
  Median1474
  Interquartile range7.7554.75
  Mean16.1186.11
  SD6.9141.87
  P-value vs. healthy volunteers (Mann-Whitney)0.1770.046
Relatives who did not inherit the familial BRCA1/2 mutation
  Median1669
  Interquartile range826
  Mean17.2368.11
  SD7.7422.30
  P-value vs. healthy volunteers (Mann-Whitney)0.1160.400

[i] SD standard deviation, MN micronucleus, BN binucleated cells.

Compared to HV without a family history of BC/OC, BRCA2 mutation carriers showed a significant increase in mean MN yields after exposure to 2 Gy IR (P=0.046; Mann-Whitney). Conversely, the radiation-induced MN yields were similar in relatives who did not inherit the familial BRCA1/2 mutation and HV without a family history of BC/OC. The mean MN yield in BRCA2 mutation carriers was higher compared to the mean yield in non-carriers (86.11 vs. 68.11 MN/1,000 BN cells, respectively). This difference, however, was not significant (P=0.298; Mann-Whitney), probably due to the small cohort and the high SD (Table I and Fig. 1). Furthermore, MN yields did not differ between non-carrier relatives from BRCA1 or BRCA2 pedigrees (Table II).

Table II.

Overview of median, interquartile range, mean and SD of the micronucleus yield (#MN/1,000 BN) for healthy relatives who did not inherit the familial germline BRCA1/2 mutation.

Table II.

Overview of median, interquartile range, mean and SD of the micronucleus yield (#MN/1,000 BN) for healthy relatives who did not inherit the familial germline BRCA1/2 mutation.

Goup data0 Gy2 Gy
Relatives who did not inherit the familial BRCA1 mutation (n=9)
  Median1466
  Interquartile range1257
  Mean16.4469.04
  SD6.8827.45
Relatives who did not inherit the familial BRCA2 mutation (n=8)
  Median1670
  Interquartile range12.7551.71
  Mean18.1166.98
  SD9.1816.59
  P-value vs. BRCA1 non-carriers (Mann-Whitney)0.73390.9601

[i] SD standard deviation, MN micronucleus, BN binucleated cells.

The individual MN yields after exposure to 2 Gy and the RS score for each BRCA2 mutation carrier, non-carrier relative and healthy volunteer group are listed in Table III. Furthermore, Table III shows mutational data (both nucleotide and protein nomenclature) and individuals with the same family ID are related. Fig. 2 shows the distribution of the 3 groups for the different RS scores. A significantly higher number of BRCA2 mutation carriers (n=9/18; 50%) showed increased RS scores (score 1 or 2) compared to HV (n=3/18; 17%) (P=0.038; one-tailed Fishers exact test). For the relatives who did not inherit the familial germline mutation only 24% (n=4/17) showed an elevated radiosensitivity at the individual level. RS scoring in related individuals (see family ID in Table III) however shows some variation. An OR of 5 (95% CI, 1.07–23.46) for BRCA2 mutation carriers vs. HV, indicates a significant association between the presence of a BRCA2 mutation and radiosensitivity according to our criteria.

Table III.

Germline mutation, family ID, micronucleus yields (#MN/1,000 BN) and RS score for BRCA2 mutation carriers, relatives who did not inherit the familial mutation (non-carrier relatives) and healthy volunteers (numbering of the nucleotides according to RefSeq nr. NM_000059.3; A of ATG start codon=nucleotide +1).

Table III.

Germline mutation, family ID, micronucleus yields (#MN/1,000 BN) and RS score for BRCA2 mutation carriers, relatives who did not inherit the familial mutation (non-carrier relatives) and healthy volunteers (numbering of the nucleotides according to RefSeq nr. NM_000059.3; A of ATG start codon=nucleotide +1).

BRCA2 mutation carriersNon-carrier relativesHealthy volunteers



IDFamily IDMutation: nucleotideMutation: protein0 Gy2 GyRS scoreIDFamily IDFamily gene0 Gy2 GyRS scoreID0 Gy2 GyRS score
M2.01BR-32-2170c.658_659delGTp.(Val220fs*4)141192NM.06BR-32-0156BRCA217510D0119831
M2.02BR-32-1748c.1389_1390delp.(Val464fs*3)15911NM.17BR-32-0342BRCA18290D1210520
M2.03BR-32-1748c.1389_1390delp.(Val464fs*3)19831NM.01BR-32-0645BRCA120630D137470
M2.04BR-32-1748c.1389_1390delp.(Val464fs*3)12580NM.10BR-32-1134BRCA114740D1512550
M2.05BR-32-1748c.1389_1390delp.(Val464fs*3)16560NM.13BR-32-1225BRCA118430D1617440
M2.06BR-32-1758c.1989delp.(Phe663fs*5)121632NM.12BR-32-1225BRCA111570D177580
M2.07BR-32-0884c.4171del p.(Glu1391fs*19)37650NM.07BR-32-1444BRCA112660D2113480
M2.08BR-32-0884c.4171del p.(Glu1391fs*19)20901NM.08BR-32-1444BRCA112780D0412400
M2.09BR-32-1759c.4936_4939del p.(Glu1646fs*23)20650NM.02BR-32-1494BRCA124871D056300
M2.10BR-32-1759c.4936_4939del p.(Glu1646fs*23)18530NM.16BR-32-1967BRCA1291252D0615740
M2.11BR-32-0156c.6275_6276del p.(Leu2092Profs*7)12630NM.03BR-32-0884BRCA220911D299290
M2.12BR-32-1565c.6275_6276del p.(Leu2092Profs*7)8440NM.04BR-32-0884BRCA216730D30301092
M2.13BR-32-1930c.6275_6276del p.(Leu2092Profs*7)231832NM.09BR-32-1748BRCA221700D327961
M2.14BR-32-1930c.6275_6276del p.(Leu2092Profs*7)12861NM.11BR-32-1758BRCA28450D3126730
M2.15BR-32-1920c.8167G>Cp.(Asp2723His)101182NM.05BR-32-1759BRCA238851D3537760
M2.16BR-32-1628 c.8332-?_8487-?del p.(Ile2778Lysfs*40)22290NM.14BR-32-1759BRCA212690D376750
M2.17BR-32-0937c.8904delC p.(Val2969fs*7)101312NM.15BR-32-2170BRCA213520D3817520
M2.18BR-32-0082c.9256+1G>C r.9118_9256del;10530 D398610
p.(Val3040Aspfs*18)
Median 14.0074.00 16.0069.00 12.0056.00
Interquartile range 7.7554.75 8.0026.00 9.7527.50
Mean 16.1186.11 17.2368.11 14.3361.22
SD 6.9141.87 7.7422.30 8.8521.73

[i] The splice site mutation present in M2.18 was previously described by Claes et al (39), where it was erroneously defined as IVS24G>A. SD standard deviation, MN micronucleus, BN binucleated cells, RS radiosensitivity.

All but one of the 18 mutation carriers enrolled in the present study were heterozygous for a mutation predicted to result in a premature termination codon (PTC). The patient with the deleterious missense mutation [BRCA2 c.8167G>C; p.(Asp2723His)] obtained an RS score of 2.

Discussion

Results of the G2 micronucleus (MN) assay performed after exposure to 2 Gy γ-rays showed a significantly increased radiosensitivity in healthy BRCA2 mutation carriers compared to healthy controls. Previous studies with a large number of different techniques were able to demonstrate enhanced radiosensitivity in BC patients with a BRCA2 mutation, however, no univocal results were achieved for healthy BRCA2 mutation carriers (2025). Non-carrier relatives of either BRCA1 or BRCA2 families did not show an increased radiosensitive phenotype compared to the cohort of healthy volunteers, which is in agreement with the study of Baeyens et al (20). We previously performed the G2 MN assay in a group of 18 healthy BRCA1 mutation carriers, and found a significantly increased MN yield after exposure to 2 Gy γ-rays (12). These findings are analogous to the results of the present study, performed in healthy carriers of pathogenic BRCA2 mutations. Fig. 3 shows the integration of the data from healthy BRCA1 mutation carriers in the present study. The detection of an increased mean MN yield in both BRCA1 and BRCA2 mutation carriers after exposure to ionizing radiation can be explained by their mutual role in DNA double-strand break repair reviewed by Roy et al (1).

In our previous study we also analyzed the G2/M checkpoint activity by the addition of caffeine, an agent abrogating the G2/M checkpoint, to the irradiated cultures and demonstrated a significantly impaired checkpoint activation in BRCA1 mutation carriers compared to healthy volunteers (12). Analysis of the G2/M checkpoint activation in the current BRCA2 cohort did not reveal a significant difference (data not shown). This result is in agreement with the fact that BRCA2 is not activated in this particular checkpoint pathway as reviewed by Roy et al (1), but does not support the data obtained by Menzel et al (28), suggesting a role for BRCA2 as a regulator of G2 checkpoint maintenance following DNA damage introduced in a human osteosarcoma cell line (U2OS) expressing dominant-negative p53 by a high-dose of ionizing radiation (6 Gy).

The role of BRCA2 in the HR pathway, a DNA repair pathway active in the S and G2 phase of the cell cycle, is extensively reported in literature (1). The present study, focusing on radiosensitivity testing of lymphocytes in these phases of the cell cycle, showed an OR of 5 (95% CI, 1.07–23.47) for healthy individuals with a heterozygous BRCA2 mutation compared to healthy controls. This indicates a positive association between the presence of a BRCA2 mutation and radiosensitivity that could be attributed to deficient HR capacity in heterozygous cells.

Two independent research groups have reported a decreased DSB repair capacity in BRCA2 heterozygous cells. Keimling et al used an enhanced green fluorescent protein (EGFP)-based assay to report impaired HR capacity in lymphoblastoid cells with a BRCA2 monoallelic truncating frameshift mutation. They confirmed this decrease in HR capacity in a BRCA2-knockdown HeLa cell line (29). Arnold et al demonstrated distinct defects in DNA DSB repair in lymphoblastoid cell lines (LCLs) from heterozygous BRCA2 mutation carriers through analysis of γ-H2AX repair kinetics (30). Although, the latter study did not focus on DNA repair by HR, it indicates a malfunction of DSB repair in LCLs from BRCA2 mutation carriers that could be attributed to diminished HR activity.

Most mutation carriers enrolled in the present study (n=17/18, 94%) had a mutation resulting in a premature termination codon (PTC). The presence of a PTC mutation is expected to activate nonsense-mediated decay of the gene transcript. Previous research from various groups including ours, demonstrated a reduction in mutant mRNA to approximately half of the WT mRNA levels in lymphocytes of individuals with a PTC mutation in BRCA1 (12,31,32). Arnold et al (30) detected a similar mutant mRNA reduction for BRCA2 mutations leading to a PTC. Furthermore, Arnold et al (30) and Keimling et al (29) report distinct reduced protein levels in LCLs from heterozygous BRCA2 mutation carriers, although quantitative analysis of this variation was not performed. Previously, haploinsufficiency has been suggested as the mechanism for hereditary BC development in BRCA1 and BRCA2 mutation carriers (33). In the present study, a higher than expected number of radiosensitive individuals in the BRCA2 mutation carriers indicates that haploinsufficiency may also be responsible for the radiosensitive phenotype in carriers of a mutation generating a PTC. In the present study, only one individual with a deleterious missense mutation was included. This substitution results in an amino acid change at position p.2723 and impairs protein functionality as shown by a homology-directed DNA break-repair functional assay (34). For this individual we obtained a high RS score of 2. Further research in larger patient cohorts with different types of mutations is needed to evaluate whether the type of mutation influences the radiosensitive phenotype or whether there are additional parameters determining this phenotype.

Results of the G2 MN assay showed no increased radiosensitivity in the group of non-carrier relatives of both BRCA1 and BRCA2 families compared to a group of healthy volunteers. Furthermore, only 24% of non-carriers showed an elevated radiosensitivity at the individual level (RS score 1 or 2). This was not significantly different from the fraction of healthy volunteers (17%) that was found to have an increased RS score. In addition, no difference was observed between non-carriers from BRCA1 (RS score, 0 in 7/9 investigated relatives) or BRCA2 families (RS score, 0 in 6/8 investigated relatives). However, we observed some variation within the different groups. We hypothesize that modifiers may play a role: indeed, selected SNPs in DNA-damage repair genes and other common variants have been associated with increased radiosensitivity (3537) and increased BC risk (35,38). Further and larger studies are needed to evaluate the subtle influence of possible modifying factors on BC risk and radiosensitivity.

In conclusion, the present study demonstrated higher radiosensitivity in healthy BRCA2 mutation carriers compared to healthy volunteers by means of the G2 MN assay after exposure of peripheral blood lymphocytes to a dose of 2 Gγ-rays. No increased radiosensitivity was observed in non-carrier relatives of BRCA1 and BRCA2 families. When evaluating radiosensitivity at the individual level, a significantly higher proportion of BRCA2 mutation carriers (50%) showed a mild or more severe radiosensitivity compared to healthy volunteers (17%) and non-carriers (24%). Furthermore, an OR of 5 indicated a positive association between the BRCA2 mutation and an increased radiosensitivity in healthy mutation carriers. These results indicate that care should be taken when applying ionizing radiation for either diagnostic or therapeutic purposes in BRCA2 mutation carriers. However, a study including a larger population of subjects carrying different types of BRCA2 mutations and non-carriers, must be performed to further elucidate the effect of each single mutation on the radiosensitive phenotype and the influence of possible underlying factors.

Acknowledgements

The authors would like to thank all participants who donated a blood sample for the present study. They thank Céline De Brock, Brecht Crombez, Ilse Coene, Johanna Aernoudt, Toke Thiron, Greet De Smet and Leen Pieters for their technical assistance. Professor Thierens is thanked for the use of the irradiation facility. The present study was funded by the Belgian Foundation against Cancer (project 2012–216).

References

1 

Roy R, Chun J and Powell SN: BRCA1 and BRCA2: Different roles in a common pathway of genome protection. Nat Rev Cancer. 12:68–78. 2011. View Article : Google Scholar : PubMed/NCBI

2 

Bernholtz S, Laitman Y, Kaufman B, Shimon-Paluch S and Friedman E: Phenocopy breast cancer rates in Israeli BRCA1 BRCA2 mutation carrier families: Is the risk increased in non-carriers? Breast Cancer Res Treat. 132:669–673. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Domchek SM, Gaudet MM, Stopfer JE, Fleischaut MH, Powers J, Kauff N, Offit K, Nathanson KL and Robson M: Breast cancer risks in individuals testing negative for a known family mutation in BRCA1 or BRCA2. Breast Cancer Res Treat. 119:409–414. 2010. View Article : Google Scholar : PubMed/NCBI

4 

Harvey SL, Milne RL, McLachlan SA, Friedlander ML, Birch KE, Weideman P, Goldgar D, Hopper JL and Phillips KA: kConFab Investigators: Prospective study of breast cancer risk for mutation negative women from BRCA1 or BRCA2 mutation positive families. Breast Cancer Res Treat. 130:1057–1061. 2011. View Article : Google Scholar : PubMed/NCBI

5 

Korde LA, Mueller CM, Loud JT, Struewing JP, Nichols K, Greene MH and Mai PL: No evidence of excess breast cancer risk among mutation-negative women from BRCA mutation-positive families. Breast Cancer Res Treat. 125:169–173. 2011. View Article : Google Scholar : PubMed/NCBI

6 

Kurian AW, Gong GD, John EM, Johnston DA, Felberg A, West DW, Miron A, Andrulis IL, Hopper JL, Knight JA, et al: Breast cancer risk for noncarriers of family-specific BRCA1 and BRCA2 mutations: Findings from the Breast Cancer Family Registry. J Clin Oncol. 29:4505–4509. 2011. View Article : Google Scholar : PubMed/NCBI

7 

Nielsen HR, Petersen J, Krogh L, Nilbert M and Skytte AB: No evidence of increased breast cancer risk for proven noncarriers from BRCA1 and BRCA2 families. Fam Cancer. 15:523–528. 2016. View Article : Google Scholar : PubMed/NCBI

8 

Smith A, Moran A, Boyd MC, Bulman M, Shenton A, Smith L, Iddenden R, Woodward ER, Lalloo F, Maher ER, et al: Phenocopies in BRCA1 and BRCA2 families: Evidence for modifier genes and implications for screening. J Med Genet. 44:10–15. 2007. View Article : Google Scholar : PubMed/NCBI

9 

Vos JR, De Bock GH, Teixeira N, van der Kolk DM, Jansen L, Mourits MJE and Oosterwijk JC: Proven non-carriers in BRCA families have an earlier age of onset of breast cancer. Eur J Cancer. 49:2101–2106. 2013. View Article : Google Scholar : PubMed/NCBI

10 

Evans DGR, Ingham SL, Buchan I, Woodward ER, Byers H, Howell A, Maher ER, Newman WG and Lalloo F: Increased rate of phenocopies in all age groups in BRCA1/BRCA2 mutation kindred, but increased prospective breast cancer risk is confined to BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev. 22:2269–2276. 2013. View Article : Google Scholar : PubMed/NCBI

11 

Pijpe A, Andrieu N, Easton DF, Kesminiene A, Cardis E, Noguès C, Gauthier-Villars M, Lasset C, Fricker JP, Peock S, et al: GENEPSO; EMBRACE; HEBON: Exposure to diagnostic radiation and risk of breast cancer among carriers of BRCA1/2 mutations: Retrospective cohort study (GENE-RAD-RISK). BMJ. 345:e56602012. View Article : Google Scholar : PubMed/NCBI

12 

Baert A, Depuydt J, Van Maerken T, Poppe B, Malfait F, Storm K, van den Ende J, Van Damme T, De Nobele S, Perletti G, et al: Increased chromosomal radiosensitivity in asymptomatic carriers of a heterozygous BRCA1 mutation. Breast Cancer Res. 18:522016. View Article : Google Scholar : PubMed/NCBI

13 

Andrieu N, Easton DF, Chang-Claude J, Rookus MA, Brohet R, Cardis E, Antoniou AC, Wagner T, Simard J, Evans G, et al: Effect of chest X-rays on the risk of breast cancer among BRCA1/2 mutation carriers in the international BRCA1/2 carrier cohort study: A report from the EMBRACE, GENEPSO, GEO-HEBON, and IBCCS Collaborators' Group. J Clin Oncol. 24:3361–3366. 2006. View Article : Google Scholar : PubMed/NCBI

14 

Lecarpentier J, Noguès C, Mouret-Fourme E, Stoppa-Lyonnet D, Lasset C, Caron O, Fricker JP, Gladieff L, Faivre L, Sobol H, et al: GENEPSO: Variation in breast cancer risk with mutation position, smoking, alcohol, and chest X-ray history, in the French National BRCA1/2 carrier cohort (GENEPSO). Breast Cancer Res Treat. 130:927–938. 2011. View Article : Google Scholar : PubMed/NCBI

15 

Narod SA, Lubinski J, Ghadirian P, Lynch HT, Moller P, Foulkes WD, Rosen B, Kim-Sing C, Isaacs C, Domchek S, et al: Hereditary Breast Cancer Clinical Study Group: Screening mammography and risk of breast cancer in BRCA1 and BRCA2 mutation carriers: A case-control study. Lancet Oncol. 7:402–406. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Giannakeas V, Lubinski J, Gronwald J, Moller P, Armel S, Lynch HT, Foulkes WD, Kim-Sing C, Singer C, Neuhausen SL, et al: Mammography screening and the risk of breast cancer in BRCA1 and BRCA2 mutation carriers: A prospective study. Breast Cancer Res Treat. 147:113–118. 2014. View Article : Google Scholar : PubMed/NCBI

17 

Goldfrank D, Chuai S, Bernstein JL, Ramon Y, Cajal T, Lee JB, Alonso MC, Diez O, Baiget M, Kauff ND, Offit K, et al: Effect of mammography on breast cancer risk in women with mutations in BRCA1 or BRCA2. Cancer Epidemiol Biomarkers Prev. 15:2311–2313. 2006. View Article : Google Scholar : PubMed/NCBI

18 

John EM, McGuire V, Thomas D, Haile R, Ozcelik H, Milne RL, Felberg A, West DW, Miron A, Knight JA, et al: Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab); Whittemore AS: Diagnostic chest X-rays and breast cancer risk before age 50 years for BRCA1 and BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev. 22:1547–1556. 2013. View Article : Google Scholar : PubMed/NCBI

19 

Bernstein JL, Thomas DC, Shore RE, Robson M, Boice JD Jr, Stovall M, Andersson M, Bernstein L, Malone KE, Reiner AS, et al: WECARE Study Collaborative Group: Contralateral breast cancer after radiotherapy among BRCA1 and BRCA2 mutation carriers: A WECARE study report. Eur J Cancer. 49:2979–2985. 2013. View Article : Google Scholar : PubMed/NCBI

20 

Baeyens A, Thierens H, Claes K, Poppe B, de Ridder L and Vral A: Chromosomal radiosensitivity in BRCA1 and BRCA2 mutation carriers. Int J Radiat Biol. 80:745–756. 2004. View Article : Google Scholar : PubMed/NCBI

21 

Gutiérrez-Enríquez S, Ramón Y, Cajal T, Alonso C, Corral A, Carrasco P, Cornet M, Sanz J, Ribas M, Baiget M and Diez O: Ionizing radiation or mitomycin-induced micronuclei in lymphocytes of BRCA1 or BRCA2 mutation carriers. Breast Cancer Res Treat. 127:611–622. 2011. View Article : Google Scholar : PubMed/NCBI

22 

Trenz K, Rothfuss A, Schütz P and Speit G: Mutagen sensitivity of peripheral blood from women carrying a BRCA1 or BRCA2 mutation. Mutat Res. 500:89–96. 2002. View Article : Google Scholar : PubMed/NCBI

23 

Beroukas E, Pandis N, Giannoukakos K, Rizou E, Beroukas K, Giatromanolaki A and Koukourakis M: Increased chromosomal radiosensitivity in women carrying BRCA1/BRCA2 mutations assessed with the G2 assay. Int J Radiat Oncol Biol Phys. 76:1199–1205. 2010. View Article : Google Scholar : PubMed/NCBI

24 

Becker AA, Graeser MK, Landwehr C, Hilger T, Baus W, Wappenschmidt B, Meindl A, Weber RG and Schmutzler RK: A 24-color metaphase-based radiation assay discriminates heterozygous BRCA2 mutation carriers from controls by chromosomal radiosensitivity. Breast Cancer Res Treat. 135:167–175. 2012. View Article : Google Scholar : PubMed/NCBI

25 

Bolognesi C, Bruzzi P, Gismondi V, Volpi S, Viassolo V, Pedemonte S and Varesco L: Clinical application of micronucleus test: A case-control study on the prediction of breast cancer risk/susceptibility. PLoS One. 9:e1123542014. View Article : Google Scholar : PubMed/NCBI

26 

Cardinale F, Bruzzi P and Bolognesi C: Role of micronucleus test in predicting breast cancer susceptibility: A systematic review and meta-analysis. Br J Cancer. 106:780–790. 2012. View Article : Google Scholar : PubMed/NCBI

27 

Claes K, Depuydt J, Taylor AMR, Last JI, Baert A, Schietecatte P, Vandersickel V, Poppe B, De Leeneer K, D'Hooghe M, et al: Variant ataxia telangiectasia: Clinical and molecular findings and evaluation of radiosensitive phenotypes in a patient and relatives. Neuromolecular Med. 15:447–457. 2013. View Article : Google Scholar : PubMed/NCBI

28 

Menzel T, Nähse-Kumpf V, Kousholt AN, Klein DK, Lund-Andersen C, Lees M, Johansen JV, Syljuåsen RG and Sørensen CS: A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance. EMBO Rep. 12:705–712. 2011. View Article : Google Scholar : PubMed/NCBI

29 

Keimling M, Volcic M, Csernok A, Wieland B, Dörk T and Wiesmüller L: Functional characterization connects individual patient mutations in ataxia telangiectasia mutated (ATM) with dysfunction of specific DNA double-strand break-repair signaling pathways. FASEB J. 25:3849–3860. 2011. View Article : Google Scholar : PubMed/NCBI

30 

Arnold K, Kim MK, Frerk K, Edler L, Savelyeva L, Schmezer P and Wiedemeyer R: Lower level of BRCA2 protein in heterozygous mutation carriers is correlated with an increase in DNA double strand breaks and an impaired DSB repair. Cancer Lett. 243:90–100. 2006. View Article : Google Scholar : PubMed/NCBI

31 

Perrin-Vidoz L, Sinilnikova OM, Stoppa-Lyonnet D, Lenoir GM and Mazoyer S: The nonsense-mediated mRNA decay pathway triggers degradation of most BRCA1 mRNAs bearing premature termination codons. Hum Mol Genet. 11:2805–2814. 2002. View Article : Google Scholar : PubMed/NCBI

32 

Anczuków O, Ware MD, Buisson M, Zetoune AB, Stoppa-Lyonnet D, Sinilnikova OM and Mazoyer S: Does the nonsense-mediated mRNA decay mechanism prevent the synthesis of truncated BRCA1, CHK2, and p53 proteins? Hum Mutat. 29:65–73. 2008. View Article : Google Scholar : PubMed/NCBI

33 

Berger AH, Knudson AG and Pandolfi PP: A continuum model for tumour suppression. Nature. 476:163–169. 2011. View Article : Google Scholar : PubMed/NCBI

34 

Guidugli L, Pankratz VS, Singh N, Thompson J, Erding CA, Engel C, Schmutzler R, Domchek S, Nathanson K, Radice P, et al: A classification model for BRCA2 DNA binding domain missense variants based on homology-directed repair activity. Cancer Res. 73:265–275. 2013. View Article : Google Scholar : PubMed/NCBI

35 

Willems P, Claes K, Baeyens A, Vandersickel V, Werbrouck J, De Ruyck K, Poppe B, van den Broecke R, Makar A, Marras E, et al: Polymorphisms in nonhomologous end-joining genes associated with breast cancer risk and chromosomal radiosensitivity. Genes Chromosomes Cancer. 47:137–148. 2008. View Article : Google Scholar : PubMed/NCBI

36 

Guo Z, Shu Y, Zhou H, Zhang W and Wang H: Radiogenomics helps to achieve personalized therapy by evaluating patient responses to radiation treatment. Carcinogenesis. 36:307–317. 2015. View Article : Google Scholar : PubMed/NCBI

37 

Popanda O, Marquardt JU, Chang-Claude J and Schmezer P: Genetic variation in normal tissue toxicity induced by ionizing radiation. Mutat Res. 667:58–69. 2009. View Article : Google Scholar : PubMed/NCBI

38 

Antoniou AC, Kuchenbaecker KB, Soucy P, Beesley J, Chen X, McGuffog L, Lee A, Barrowdale D, Healey S, Sinilnikova OM and et al; CIMBA: SWE-BRCA; HEBON; EMBRACE; GEMO Collaborators Study; kConFab Investigators: Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. Breast Cancer Res. 14:R332012. View Article : Google Scholar : PubMed/NCBI

39 

Claes K, Poppe B, Machackova E, Coene I, Foretova L, DePaepe A and Messiaen L: Differentiating pathogenic mutations from polymorphic alterations in the splice sites of BRCA1 and BRCA2. Genes Chromosomes Cancer. 37:314–320. 2003. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

March 2017
Volume 37 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Baert, A., Depuydt, J., Van Maerken, T., Poppe, B., Malfait, F., Van Damme, T. ... Vral, A. (2017). Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay. Oncology Reports, 37, 1379-1386. https://doi.org/10.3892/or.2017.5407
MLA
Baert, A., Depuydt, J., Van Maerken, T., Poppe, B., Malfait, F., Van Damme, T., De Nobele, S., Perletti, G., De Leeneer, K., Claes, K. B., Vral, A."Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay". Oncology Reports 37.3 (2017): 1379-1386.
Chicago
Baert, A., Depuydt, J., Van Maerken, T., Poppe, B., Malfait, F., Van Damme, T., De Nobele, S., Perletti, G., De Leeneer, K., Claes, K. B., Vral, A."Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay". Oncology Reports 37, no. 3 (2017): 1379-1386. https://doi.org/10.3892/or.2017.5407