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Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma

Abstract

Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.

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Figure 1: Schematic showing the distribution of MED12 exon 2 mutations.
Figure 2: Gene expression analysis of fibroadenoma tumors.

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Gene Expression Omnibus

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Acknowledgements

This work was supported in part by funding from the Singapore National Medical Research Council (NMRC/STAR/0006/2009), the Singapore Millennium Foundation, the Lee Foundation, the Tanoto Foundation, the Singapore National Cancer Centre Research Fund, the Duke-NUS Graduate Medical School, the Cancer Science Institute, Singapore and the Verdant Foundation, Hong Kong. We thank the Duke-NUS Genome Biology Facility for sequencing services rendered, as well as the Advanced Molecular Pathology Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore for providing LCM resources. We also thank the SingHealth Tissue Repository for frozen tissue samples.

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Authors

Contributions

W.K.L., C.K.O., J.T., S.G.R., P.H.T., P.T. and B.T.T. conceived the study. S.G.R., P.H.T., P.T. and B.T.T. directed the study. J.R.M., W.K.L., I.C., S.N. and G.P. performed the bioinformatics analysis. A.A.T., W.S.O., V.K.M.T., M.H., K.W.O., B.K.T.T. and P.H.T. were involved in the procurement and histopathological review of the samples. S.S.M. was involved in specimen collection and preparation. C.C.Y.N., V.R. and S.T.T. performed whole-exome, amplicon and Sanger sequencing. N.D.M.N. performed laser capture microdissection. W.K.L., C.K.O., J.T., S.G.R., P.H.T., P.T. and B.T.T. wrote the manuscript with the assistance and final approval of all authors.

Corresponding authors

Correspondence to Steven G Rozen or Puay Hoon Tan or Patrick Tan or Bin Tean Teh.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Confirmation of somatic MED12 mutations in fresh frozen fibroadenoma samples by Sanger sequencing.

Genomic DNA Sanger sequencing of MED12 variants in eight fresh frozen fibroadenomas and their matched whole-blood. Variant allele frequency as determined by ultra-deep targeted amplicon sequencing is provided on the left of each sample.

Supplementary Figure 2 Sanger sequencing of MED12 using cDNA from fresh frozen fibroadenomas confirms transcription of mutant MED12.

Complementary DNA Sanger sequencing of MED12 variants in eight fresh frozen fibroadenomas and their matched whole-blood. Variant peaks were unambiguous except for Sample002, possibly due to RNA degradation (the sample had a RNA integrity number of 7.3; the lowest among eight samples included in our microarray study). Other explanations include the mutant allele simply not being expressed, or PCR bias towards the wild-type allele.

Supplementary Figure 3 Laser capture microdissection of Sample004.

Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample004. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 aberrant splice site mutation is exclusive to the stromal compartment.

Supplementary Figure 4 Laser capture microdissection of Sample006.

Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample006. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.

Supplementary Figure 5 Laser capture microdissection of Sample007.

Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample007. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.

Supplementary Figure 6 Laser capture microdissection of Sample008.

Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample008. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.

Supplementary Figure 7 GSEA analysis of genes upregulated in fibroadenoma and UL.

GSEA analysis similar to Fig. 2b, but against genes upregulated 2x in UL instead of 4x.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-7 and Supplementary Tables 2, 5 and 7 (PDF 18985 kb)

Supplementary Table 1

Clinical characteristics of fibroadenoma patients. (XLS 38 kb)

Supplementary Table 3

List of confirmed somatic mutations identified from whole-exome sequencing of eight fibroadenomas. (XLS 35 kb)

Supplementary Table 4

Mutations detected in ultra-deep targeted amplicon sequencing of MED12 exon 2 in 98 fibroadenoma samples. (XLS 36 kb)

Supplementary Table 6

Top 50 enriched MSigDB curated (c2) gene sets for upregulated and downregulated genes in MED12 mutant fibroadenomas. Gene sets of interest are highlighted. ES: Enrichment Score, NES: Normalized Enrichment Score, FDR: False Discovery Rate (XLS 42 kb)

Supplementary Table 8

Differentially expressed genes between mutant and wild-type MED12 fibroadenoma samples. (XLS 38 kb)

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Lim, W., Ong, C., Tan, J. et al. Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma. Nat Genet 46, 877–880 (2014). https://doi.org/10.1038/ng.3037

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