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Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors

Abstract

High rates of APOBEC-signature mutations are found in many tumors, but factors affecting this mutation pattern are not well understood. Here we explored the contribution of two common germline variants in the APOBEC3 region. SNP rs1014971 was associated with bladder cancer risk, increased APOBEC3B expression, and enrichment with APOBEC-signature mutations in bladder tumors. In contrast, a 30-kb deletion that eliminates APOBEC3B and creates an APOBEC3AAPOBEC3B chimera was not important in bladder cancer, whereas it was associated with breast cancer risk and enrichment with APOBEC-signature mutations in breast tumors. In vitro, APOBEC3B expression was predominantly induced by treatment with a DNA-damaging drug in bladder cancer cell lines, and APOBEC3A expression was induced as part of the antiviral interferon-stimulated response in breast cancer cell lines. These findings suggest a tissue-specific role of environmental oncogenic triggers, particularly in individuals with germline APOBEC3 risk variants.

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Figure 1: Fine-mapping analysis of the 22q13.1 region for association with bladder cancer risk.
Figure 2: Analysis of factors contributing to APOBEC mutagenesis in bladder and breast tumors in TCGA.
Figure 3: In silico and experimental analysis of the 2-kb region that includes GWAS SNP rs1014971 and its two proxy SNPs (r2 ≥ 0.8 in Europeans).
Figure 4: Expression of APOBEC3 genes in HT-1376 bladder and MCF-7 breast cancer cell lines infected with Sendai virus or treated with the DNA-damaging drug bleomycin.
Figure 5: Overall survival of TCGA patients with bladder cancer is improved with increased APOBEC mutagenesis and in carriers of the AA bladder cancer risk genotype at rs17000526.
Figure 6: APOBEC mutagenesis in cancer.

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Acknowledgements

We thank the Breast Cancer Association Consortium (BCAC) for access to summary results for the association between rs1014971 and breast cancer risk. The results presented here are in part based upon data generated by the TCGA Research Network. The study was supported by federal funds from the Intramural Research Program (IRP), Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health (contract HHSN261200800001E). The Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial was funded with National Institutes of Health Genes, Environment, and Health Initiative (GEI) grants HG-06-033-NCI-01 and RO1HL091172-01, U01HG004438, and NIH HHSN268200782096C. The Spanish Bladder Cancer Study (SBCS) was funded with intramural contract NCI N02-CP-11015. FIS/Spain 98/1274, FIS/Spain 00/0745, PI061614, and G03/174, Fundació Marató TV3, Red Temática Investigación Cooperativa en Cáncer (RTICC), Consolíder ONCOBIO, EU-FP7-201663; and RO1-CA089715 and CA34627. The Biobank Japan Project was supported by the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government. The funders did not have a role in study design, data collection and analysis, writing, or submission of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

L.P.-O., A.R.B., and C.D.M. designed the study and performed genetic and molecular analysis of public and laboratory-generated data. A.R.B., K.M., K.-I.U., and A.P. performed genotyping of germline variants. A.R.B., K.-I.U., and O.O.O. performed functional experiments. B.Z. contributed analytical tools. J.D.F., S.K., M. Kubo, T.S., N.D.F., M. Kogevinas, N.M., S.J.C., M.G.-C., D.T.S., and N.R. contributed samples and data. L.P.-O., A.R.B., and C.D.M. wrote the manuscript. All authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to Ludmila Prokunina-Olsson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 LD (r2) between the A3AB deletion and all markers in a 400-kb APOBEC3 region in 1000 Genomes Project populations.

Populations: CEU, individuals of European ancestry from Utah, only samples that overlapped with the HapMap set are used here; CHB, Chinese from Beijing; JPT, Japanese from Tokyo. All samples were genotyped with a CNV assay; genotypes for all other markers were generated by the 1000 Genomes Project (October 2014 release). SNP rs12628403 is the only marker that tags the deletion in Europeans and Japanese (r2 = 1.0) and Chinese (r2 = 0.95). In the Yoruba (YRI) panel of the 1000 Genomes Project, the CNV is weakly polymorphic (4.2%) while rs12628403 is monomorphic, and LD metrics could not be calculated. SNP rs12628403 was also genotyped by a custom TaqMan assay (Supplementary Note), and all genotypes were 100% concordant with data from the 1000 Genomes Project.

Supplementary Figure 2 LD (r2) between the A3AB deletion, its proxy SNP rs12628403, and all GWAS-genotyped and imputed markers within a 400-kb APOBEC3 genomic region on chromosome 22q13.1.

The plot is based on 848 genotyped or imputed markers in 1,837 samples from individuals of European ancestry from the PLCO study in which the deletion (gray box) was genotyped by a CNV assay and its proxy SNP, rs12628403, was genotyped by a TaqMan genotyping assay. In this set, the CNV and rs12628403 have r2 = 0.92 and D′ = 0.97. Because of low LD with other markers in this region (best r2 ~0.2), the deletion and its proxy SNP, rs12628403, cannot be imputed and have to be genotyped.

Supplementary Figure 3 Electrophoretic mobility shift assays for SNP rs1014971 with nuclear extracts from bladder cancer cell line HTB-9 and breast cancer cell lines MDA-MB-231 and T-47D.

Supplementary Figure 4 Expression of selected APOBEC3 genes (A3A, A3B, and A3G) in GTEx.

Expression analysis in 8,555 samples (53 normal human tissues from 544 donors) based on data generated by the Genotype-Tissue Expression (GTEx) Project. Expression is measured by RNA–seq and presented as normalized log10 (FPKM) values. Expression in bladder and breast tissue samples is marked by red boxes. Data for colon–transverse tissue were available only for A3A and are labeled separately, while data for expression of A3A were not available for adipose–visceral tissue.

Supplementary Figure 5 Expression of selected APOBEC3 genes (A3A, A3B, and A3G) in bladder cancer cell lines RT-4 and HTB-9 infected with Sendai virus (SeV) or treated with the DNA-damaging drug bleomycin (Bleo).

(a,c) Increase in expression of a viral-specific RNA shows that cells were successfully infected with SeV. (b,d) In untreated cells, baseline expression of A3A is significantly lower than that of A3B and A3G. (e,g) A3A and A3G but not A3B are significantly induced after 12 h of SeV infection. (f,h) Expression of A3A, A3B, and A3G is significantly induced by 24 h of treatment with bleomycin as compared to untreated (UT) samples. Plots present expression values (ΔCt, log2 scale) for targets (A3A, A3B, and A3G) normalized by the geometric mean of expression for two endogenous controls (GAPDH and PPIA). Dotted lines indicate the lower level of detection for the targets—a Ct value of 40 was assigned to samples for which expression was not detected by 40 cycles of qRT–PCR; individual plot points for these samples are defined by the levels of expression of endogenous controls. All experiments were performed in biological triplicate. P values are for two-sided t tests. Shown are values for individual replicates and means. Raw data are available in Supplementary Data 2.

Supplementary Figure 6 Expression of selected APOBEC3 genes (A3A, A3B, and A3G) in breast cancer cell lines MDA-MB-231 and T-47D infected with Sendai virus or treated with the DNA-damaging drug bleomycin.

(a,c) Increase in expression of a viral-specific RNA shows that cells were successfully infected with SeV. (b,d) In untreated cells, A3B expression is significantly higher than that of A3A and A3G. (e,g) Only A3A in MDA-MB-231 cells and all APOBEC genes in T-47D cells are significantly induced after 12 h of SeV infection. (f,h) Only A3B and A3G in MDA-MB-231 cells and all APOBEC genes in T-47D cells are significantly induced by 24 h of treatment with bleomycin as compared to untreated (UT) samples. Plots present expression values (ΔCt, log2 scale) for targets (A3A, A3B, and A3G) normalized by the geometric mean of expression for two endogenous controls (GAPDH and PPIA). Dotted lines indicate the lower level of detection for the targets—a Ct value of 40 was assigned to samples for which expression was not detected by 40 cycles of qRT–PCR; individual plot points for these samples are defined by the level of expression of endogenous controls. All experiments were performed in biological quadruplicate. P values are for two-sided t tests. Shown are values for individual replicates and means. NE, not expressed in all samples. Raw data are available in Supplementary Data 2.

Supplementary Figure 7 APOBEC mutagenesis and SNP rs17000526 as predictors of overall survival for patients with breast cancer in TCGA.

(ah) Results are presented separately for patients with ER+ (ad) and ER (eh) tumors. (a,e) Overall survival in relation to quartiles of APOBEC-signature mutation counts. (b,f) Overall survival in relation to APOBEC mutagenesis pattern classified as “no” or “yes” (at least one mutation present). (c,g) Overall survival in relation to APOBEC mutagenesis pattern classified as “no,” “low mutation counts” (1–48 mutations) or “high mutation counts” (≥49 mutations; based on the median in bladder tumors, presented in Fig. 5b). (d,h) Overall survival in relation to rs17000526. Hazards ratios and P values are for multivariate Cox regression models that also include age and tumor stage as core variables.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 3–20 and Supplementary Note. (PDF 3510 kb)

Supplementary Table 1

Association with bladder cancer risk for top genotyped or imputed markers within 1 Mb of the 22q13.1 region. (XLSX 22 kb)

Supplementary Table 2

Genotypes of A3AB deletion (CNV) and SNP rs12628403 in HapMap populations. (XLSX 53 kb)

Supplementary Data 1

Data source for analyses in TCGA bladder tumors. Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors. (XLSX 1214 kb)

Supplementary Data 2

Source file for analyses. Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors. (XLSX 301 kb)

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Middlebrooks, C., Banday, A., Matsuda, K. et al. Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors. Nat Genet 48, 1330–1338 (2016). https://doi.org/10.1038/ng.3670

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