Abstract
Copy number variations occur frequently in the genome and are a significant source of human genetic variation accounting for disease. Recently, we discovered a common deletion located in the APOBEC3A and APOBEC3B genes significantly associated with breast cancer in Chinese women. Investigating this locus in other populations would be an expedient way to evaluate the generalizability of the novel finding. We analyzed the APOBEC3 deletion in a large study of 3273 European-ancestry women (including 1671 breast cancer cases and 1602 controls) from the population-based Nashville Breast Health Study. All participants were genotyped using real-time qualitative PCR. Logistic regression was used to derive odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between the deletion polymorphism and breast cancer risk. The APOBEC3 deletion was observed in 12.4% of cases and 10.4% of controls. The deletion was significantly associated with breast cancer risk, with ORs and 95% CIs of 1.21 (1.02–1.43) associated with one-copy deletion and 2.29 (1.04–5.06) associated with two-copy deletion compared with women with no deletion (P for trend = 0.005). The positive association of the APOBEC3 deletion with breast cancer risk was similar for estrogen receptor-positive and estrogen receptor-negative breast cancer and was not modified by known breast cancer risk factors. Results from this study confirmed the association of the APOBEC3 deletion with breast cancer risk among women of European ancestry.
Introduction
Breast cancer is the most commonly diagnosed malignancy among women in the USA. Genetic factors play an important role in the etiology of both sporadic and familial breast cancer. Recent genome-wide association studies focusing on evaluating single-nucleotide polymorphisms have identified ~67 common genetic susceptibility loci for breast cancer (1–3). However, these newly identified genetic factors, along with known high-penetrance breast cancer susceptibility genes, explain a small portion of the heritability for this cancer (4). Recently, studies have shown that copy number variations (CNV), another type of genetic variation, occur frequently in the genome and account for more nucleotide sequence variation than single-nucleotide polymorphisms (5). It has been proposed that CNVs may explain some of the missing heritability for complex traits after the genome-wide association studies (6). Recently, through CNV genome-wide association studies, we discovered a common CNV locus for breast cancer in Chinese women (7). This CNV is a deletion located between exon 5 of APOBEC3A and exon 8 of APOBEC3B, resulting in a fusion gene. This deletion is very common in East Asians with frequency of >30% but less common in European-ancestry populations with frequency of ~10% based on the 1000 Genomes Project data (http://www.1000genomes.org/). In this study, we evaluated the association of this CNV locus with breast cancer in a European-ancestry population of 1671 cases and 1602 controls to assess the generalizability of the novel finding.
Subjects and methods
Study populations
The Nashville Breast Health Study (NBHS) is a population-based case–control study of breast cancer conducted in the Nashville metropolitan area. Through a rapid case ascertainment system, we identified newly diagnosed breast cancer cases through the Tennessee State Cancer Registry and five major hospitals in the city that provide medical care for breast cancer patients. Eligible cases were women diagnosed with primary breast cancer between 1 February 2001 and 31 December 2008, who were between the ages of 25 and 75, who had no history of cancer other than non-melanoma skin cancer, who had a residential telephone, who spoke English and who were able to provide consent to the study. Controls were identified via random digit dialing of households in the same geographic area as cases. Eligibility criteria for controls were the same as cases with the exception that controls did not have a prior cancer diagnosis other than simple skin cancer. Information on the estrogen receptor (ER) and progesterone receptor (PR) status of breast cancer tumors was obtained from pathology records, and 70% of the cases had information on both ER and PR status. Controls were frequency matched to cases on 5-year age group, race and county of residence. The majority of the participants were from three counties, Davidson, Rutherford and Williamson. Information on demographic factors, as well as known and suspected risk factors for breast cancer, was ascertained through a structured questionnaire administered via telephone interview. Among 7769 eligible women, a total of 5131 women (66.0%) completed the interview. Reasons for non-participation included refusal (N = 2170), death (N = 211), illness (N = 20), unable to be reached (N = 1) and only completing a portion of the interview (N = 236). Among the 2738 cases who completed the interview, 2520 provided biological samples (92.0%); among the 2393 controls who completed the interview, 2208 provided biological samples (92.3%). Buccal cell samples were collected via two methods: Oragene saliva collection kits (DNA GenoteK, Ottawa, Canada) and mouthwash samples. Included in the current project are 1671 cases and 1602 controls of European ancestry who participated in the study before August 2008 and with biological samples available. Approval for this study was obtained from the Institutional Review Boards of Vanderbilt University Medical Center and of the individual collaborating institutions. All participants provided informed consent prior to enrollment in this study.
Copy number analyses
Copy number analyses for all participants were conducted using real-time qualitative PCR. Primers and probes highly specific to the target gene, APOBEC3B (Assay ID: Hs04504055_cn), and the reference gene, RNase P, were purchased from Applied Biosystems (ABI). The Coriell DNA NA18635, which carried two copies of the APOBEC3B gene, was used as the calibrator. The ΔΔCt was calculated by the formula: (Ct reference genesample − Ct target genesample) – (Ct reference genecalibrator − Ct target genecalibrator) (8). If there was no PCR amplification for the APOBEC3B gene after 45 cycles, while the RNase P gene was successfully amplified, the ΔΔCt value could not be estimated and these participants were determined to carry two-copy deletions of the APOBEC3B gene. When the ΔΔCt was estimated, the APOBEC3B gene was called as two-copy deletion (2X2ΔΔCt < 0.2), one-copy deletion (0.8 < 2X2ΔΔCt < 1.2) and no deletion (2X2ΔΔCt > 1.8). If the 2X2ΔΔCt value was not within the above ranges, these participants were repeated with triplicates scattered on another 384 well plate. Each 96 well plate included one negative control (water) and two samples from the HapMap project as quality control samples. Approximately, 5% of the study samples were blindly duplicated and scattered on each 96 well plate. The mean concordance was 99.6% for the blind duplicates and 98.9% for HapMap samples.
Statistical analyses
Associations between the CNV and breast cancer risk were assessed using odds ratios (ORs) and 95% confidence intervals (CIs) derived from logistic regression models. ORs were estimated for one-copy and two-copy deletion genotypes compared with no deletion genotype. The OR (95% CI) was also estimated for per copy loss based on a log-additive model and adjusted for age. Additional adjustment for education, menarche age, body mass index (BMI), county of residence and family history of breast cancer were also performed, and the results did not change materially. Analyses stratified by menopausal status, ER status, PR status, BMI, menarche age, family history of breast cancer, age and age of first live birth were carried out. Multiplicative interactions between CNV and demographic variables were evaluated using the likelihood ratio test when interaction terms were added to logistic regression models along with the main effect terms. SAS 9.2 (Cary, NC) was used to conduct these analyses.
Results
Characteristics of the study population are shown in Table I. Cases were slightly older than controls. Cases achieved less educational attainment and were from lower income households. They were more likely to have a first-degree relative with breast cancer, a history of benign breast disease, be postmenopausal and report less regular physical activity than controls.
Table I.
Distribution of demographic characteristics and known breast cancer risk factors for cases and controls, Nashville Breast Health Study 2001–2008
| Category | Cases (N = 1671) | Controls (N = 1602) | P value |
|---|---|---|---|
| Demographic factorsa | |||
| Age (year) | 54.8±10.2 | 52.9±10.8 | <0.01 |
| Education ≥ college (%) | 64.2 | 68.0 | 0.02 |
| Reporting regular exercise (%) | 53.9 | 58.2 | 0.01 |
| County of residence (%) | |||
| Davidson | 34.9 | 39.9 | <0.01 |
| Rutherford | 13.7 | 18.9 | |
| Williamson | 13.5 | 16.2 | |
| Others | 37.9 | 25.0 | |
| Reproductive risk factors | |||
| Age at menarche (year) | 12.6±2.6 | 12.8±4.0 | 0.07 |
| Postmenopausal (%)b | 66.0 | 59.3 | <0.01 |
| Age at menopauseb | 45.4±7.9 | 45.1±8.1 | 0.28 |
| Number of live birth (year)c | 2.3±1.2 | 2.3±1.0 | 0.70 |
| Age at first live birth (year)c | 20.2±10.5 | 20.5±10.4 | 0.45 |
| Other risk factors | |||
| Ever diagnosed with benign breast disease (%) | 49.1 | 33.6 | <0.01 |
| First-degree relative with breast cancer (%) | 19.9 | 14.2 | <0.01 |
| BMI | 26.98±5.99 | 26.81±6.24 | 0.42 |
| BMIb | 27.29±6.02 | 27.44±6.08 | 0.58 |
aUnless otherwise specified, mean ± SD are presented.
bAmong postmenopausal women.
cAmong parous women.
A highly significant association was observed between the APOBEC3 deletion and breast cancer risk, with ORs (95% CIs) of 1.21 (1.02–1.43) associated with one-copy deletion and 2.29 (1.04–5.06) associated with two-copy deletion compared with women with no deletion (P for trend = 5.3×10–3; Table II). After adjusting for additional potentially confounding factors, such as BMI, menarche age, educational attainment, county of residence and family history of breast cancer, the results did not change materially. The ORs (95% CIs) were 1.21 (1.02–1.43) associated with one-copy deletion and 2.57 (1.12–5.90) associated with two-copy deletion compared with women with no deletion (P = 4.6 × 10–3; Table II).
Table II.
Association between the APOBEC3 gene deletion and breast cancer risk, results from the Nashville Breast Cancer Genetics Study
| Genotypes | No. of cases | No. of controls | OR (95% CI)a | OR (95% CI)b |
|---|---|---|---|---|
| Per copy deletion | 1671 | 1602 | 1.25 (1.07–1.46) | 1.26 (1.07–1.48) |
| No deletion | 1275 | 1279 | 1.00 (reference) | 1.00 (reference) |
| One-copy deletion | 376 | 314 | 1.21 (1.02–1.43) | 1.21 (1.02–1.43) |
| Two-copy deletion | 20 | 9 | 2.29 (1.04–5.06) | 2.57 (1.12–5.90) |
| P for trend | 5.3×10–3 | 4.6×10–3 |
aAdjusted for age.
bAdjusted for age, education, BMI, age at menarche, family history of breast cancer and county of residence.
The positive association of the APOBEC3 deletion with breast cancer risk was similar when stratified by menopausal or ER or PR status. Although the association was stronger among postmenopausal women than among premenopausal women, the heterogeneity test was not statistically significant (Table III). Among women with low BMI, the association was stronger with OR (95% CI) being 1.60 (1.12–2.31) than that among women with higher BMI with an OR (95% CI) of 1.22 (0.94–1.60) (Table III). However, the interaction test was not significant. The statistical power for interaction was limited, only 50.2% to detect an interaction effect with OR being 1.25. Similarly, no significant interaction under the multiplicative model was observed between the APOBEC3 deletion and other breast cancer risk factors, such as age, age of first live birth and family history of breast cancer.
Table III.
Association of the APOBEC3 gene deletion and breast cancer stratified by breast cancer risk factorsa
| Category | Without deletion | With deletion | P for heterogeneity | ||
|---|---|---|---|---|---|
| No. of cases/no. of controls | OR (95% CI) | No. of cases/no. of controls | OR (95% CI) | ||
| Menopausal status | |||||
| Premenopausal | 435/509 | 1.00 (reference) | 133/142 | 1.08 (0.82–1.42) | 0.21 |
| Postmenopausal | 839/770 | 1.00 (reference) | 263/179 | 1.35 (1.09–1.67) | |
| ER status | |||||
| Positive cases versus controls | 779/1279 | 1.00 (reference) | 240/323 | 1.22 (1.01–1.48) | 0.88 |
| Negative cases versus controls | 242/1279 | 1.00 (reference) | 77/323 | 1.26 (0.95–1.68) | |
| PR status | |||||
| Positive cases versus controls | 649/1279 | 1.00 (reference) | 191/323 | 1.17 (0.96–1.44) | 0.32 |
| Negative cases versus controls | 362/1279 | 1.00 (reference) | 122/323 | 1.34 (1.06–1.71) | |
| Age | |||||
| ≥Median | 696/620 | 1.00 (reference) | 199/147 | 1.21 (0.95–1.53) | 0.79 |
| <Median | 579/659 | 1.00 (reference) | 197/176 | 1.26 (1.00–1.59) | |
| BMIb | |||||
| ≥25 | 503/447 | 1.00 (reference) | 168/122 | 1.22 (0.94–1.60) | 0.23 |
| <25 | 336/323 | 1.00 (reference) | 95/57 | 1.60 (1.12–2.31) | |
| Menarche age | |||||
| ≥Median | 646/676 | 1.00 (reference) | 211/163 | 1.37 (1.08–1.73) | 0.24 |
| <Median | 629/603 | 1.00 (reference) | 185/160 | 1.11 (0.88–1.42) | |
| Age at first live birth | |||||
| ≥Median | 681/710 | 1.00 (reference) | 207/185 | 1.18 (0.94–1.47) | 0.48 |
| <Median | 594/569 | 1.00 (reference) | 189/138 | 1.32 (1.03–1.69) | |
| Family history of breast cancer | |||||
| Yes | 257/175 | 1.00 (reference) | 75/53 | 0.99 (0.66–1.48) | 0.22 |
| No | 1018/1104 | 1.00 (reference) | 321/270 | 1.29 (1.08–1.55) | |
aAdjusted for age.
bAmong postmenopausal women.
Discussion
In this study, we found that the APOBEC3 deletion was significantly associated with increased risk of breast cancer among European-ancestry women. The association was similar when stratified by tumor subtype defined by ER–PR status or other breast cancer risk factors. This result is consistent with our findings in Chinese women (7) and provides additional evidence to implicate APOBEC3 deletion as a novel susceptibility factor for breast cancer.
The APOBEC3 gene family encodes proteins that play pivotal roles in intracellular defense against viral infection (9). The APOBEC3A gene has been shown to efficiently deaminate 5-methyl-dC to dT which, coupled with less efficient mismatch repair than uracil excision, could go towards explaining 5-methylcytidine mutation hotspots in cancer genomes (9). Furthermore, the APOBEC3 genes may play a role in carcinogenesis by triggering DNA mutation through dC deamination (10). The APOBEC3 gene families are expressed in most types of cells and tissues including breast cancer (11). APOBEC3B is overexpressed in many types of tumor tissues and several lymphoma cells (12,13). A recent study analyzing somatic mutations in breast cancer revealed base substitutions preferable to cytosine at TpC dinucleotides, which is a typical site targeted by APOBEC3s (14). Very recently, Roberts et al. (15) reported that mutations in C- or G-coordinated clusters in human cancer often fell into motifs of the APOBEC gene family, again indicating that APOBEC plays an important role in carcinogenesis.
The deletion is 29.5 kb in length, located between exon 5 of APOBEC3A gene and exon 8 of APOBEC3B gene. This deletion results in complete removal of the coding region of the APOBEC3B gene and is associated with decreased expression of the APOBEC3B gene in lymphoblastoid cell lines (16) and breast cancer cells (17). Somatic deletion of this 29.5kb has also been observed in breast and oral cancer tumor tissue (17) and in oligodendroglial tumors (18). This deletion has been suggested to be associated with increased risk of other diseases and conditions, including HIV-1 infection and its progression to AIDS (19) and autism (20). Recently, this deletion was also reported to be associated with falciparum malaria (21), susceptibility to persistent HBV infection and hepatocellular carcinoma (22).
There are some limitations in this study. The allele frequency is much lower in European-ancestry population than that in Chinese women, with deletion allele frequency of ~10 versus 36%. The positive association of this deletion with breast cancer risk was slightly stronger among postmenopausal women than among premenopausal women and was stronger in PR-negative tumor than in PR-positive tumor; however, the interaction test was not significant. The statistical power of this study to evaluate the interaction between this deletion and known breast cancer risk factors is low. Further functional research in this APOBEC3 region may elucidate the underlying biological mechanism of this locus that alters breast cancer risk.
In summary, we replicated the strong association between the APOBEC3 deletion with breast cancer risk for the first time in a large population of European ancestry. Further research into the function of APOBEC3 deletion and its potential biological mechanism association may be warranted.
Funding
US National Institutes of Health (R01CA137013 to J.L. and R01CA100374 to W.Z.).
Conflict of Interest Statement: None declared.
Acknowledgements
The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agents. The authors thank the study participants and research staff for their contributions and commitment to this project, Drs Alecia Fair and Alicia Beeghly-Fadiel for their contribution to the Nashville Breast Health Study, Regina Courtney for DNA preparation and Bethanie Rammer and Mary Jo Daly for assistance in the preparation of this article. Study recruitment, telephone interviews, sample preparation and CNV analyses were conducted at the Survey and Biospecimen Shared Resource and Vanderbilt Microarray Shared Resource, which are supported in part by the Vanderbilt-Ingram Cancer Center (P30 CA68485).
Glossary
Abbreviations:
- BMI
body mass index
- CI
confidence intervals
- CNV
copy number variations
- ER
estrogen receptor
- OR
odds ratios
- PR
progesterone receptor.
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