Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma

Journal name:
Nature Genetics
Volume:
46,
Pages:
877–880
Year published:
DOI:
doi:10.1038/ng.3037
Received
Accepted
Published online

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.

At a glance

Figures

  1. Schematic showing the distribution of MED12 exon 2 mutations.
    Figure 1: Schematic showing the distribution of MED12 exon 2 mutations.

    An overview of MED12 is shown with high-confidence Pfam protein domains. A close-up view of residues in MED12 exon 2 indicates the location of MED12 alterations found in this study. The frequency of each alteration is denoted in parentheses after its label. A strong preference for p.Gly44 substitutions can be observed. aa, amino acid.

  2. Gene expression analysis of fibroadenoma tumors.
    Figure 2: Gene expression analysis of fibroadenoma tumors.

    (a) Differential activation of breast cancer and estrogen signaling gene sets is associated with MED12 alterations in fibroadenomas. The heat map shows unsupervised clustering of gene sets with significantly differential activation scores as determined by GSVA. (b) GSEA enrichment plot. Genes were rank ordered according to their fold change between fibroadenoma samples with mutant MED12 and those with wild-type MED12 (bottom). Genes upregulated >4× in UL are indicated as black bars in the middle. A strong bias for these genes to also be upregulated in fibroadenoma is reflected in the top plot, which shows a running enrichment score that peaks at the extreme left of the plot.

  3. Confirmation of somatic MED12 mutations in fresh frozen fibroadenoma samples by Sanger sequencing.
    Supplementary Fig. 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.

  4. Sanger sequencing of MED12 using cDNA from fresh frozen fibroadenomas confirms transcription of mutant MED12.
    Supplementary Fig. 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.

  5. Laser capture microdissection of Sample004.
    Supplementary Fig. 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.

  6. Laser capture microdissection of Sample006.
    Supplementary Fig. 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.

  7. Laser capture microdissection of Sample007.
    Supplementary Fig. 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.

  8. Laser capture microdissection of Sample008.
    Supplementary Fig. 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.

  9. GSEA analysis of genes upregulated in fibroadenoma and UL.
    Supplementary Fig. 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.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Weng Khong Lim,
    • Choon Kiat Ong &
    • Jing Tan

Affiliations

  1. Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, Singapore.

    • Weng Khong Lim,
    • Choon Kiat Ong,
    • Jing Tan,
    • Cedric Chuan Young Ng,
    • Vikneswari Rajasegaran,
    • Swe Swe Myint,
    • Sanjanaa Nagarajan &
    • Bin Tean Teh
  2. Division of Cancer and Stem Cell Biology, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore.

    • Weng Khong Lim,
    • Choon Kiat Ong,
    • Jing Tan,
    • Cedric Chuan Young Ng,
    • Vikneswari Rajasegaran,
    • Swe Swe Myint,
    • Sanjanaa Nagarajan,
    • Su Ting Tay,
    • Patrick Tan &
    • Bin Tean Teh
  3. Department of Pathology, Singapore General Hospital, Singapore.

    • Aye Aye Thike,
    • Nur Diyana Md Nasir &
    • Puay Hoon Tan
  4. Division of Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore.

    • John R McPherson,
    • Ioana Cutcutache &
    • Steven G Rozen
  5. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

    • Gregory Poore
  6. Department of Medical Oncology, National Cancer Centre Singapore, Singapore.

    • Wei Siong Ooi
  7. Department of Surgical Oncology, National Cancer Centre Singapore, Singapore.

    • Veronique Kiak Mien Tan &
    • Kong Wee Ong
  8. Department of Surgery, National University Hospital, Singapore.

    • Mikael Hartman
  9. Department of General Surgery, Singapore General Hospital, Singapore.

    • Benita K T Tan
  10. Genome Institute of Singapore, Singapore.

    • Patrick Tan
  11. Cancer Science Institute of Singapore, National University of Singapore, Singapore.

    • Patrick Tan &
    • Bin Tean Teh

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Confirmation of somatic MED12 mutations in fresh frozen fibroadenoma samples by Sanger sequencing. (635 KB)

    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.

  2. Supplementary Figure 2: Sanger sequencing of MED12 using cDNA from fresh frozen fibroadenomas confirms transcription of mutant MED12. (646 KB)

    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.

  3. Supplementary Figure 3: Laser capture microdissection of Sample004. (327 KB)

    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.

  4. Supplementary Figure 4: Laser capture microdissection of Sample006. (355 KB)

    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.

  5. Supplementary Figure 5: Laser capture microdissection of Sample007. (368 KB)

    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.

  6. Supplementary Figure 6: Laser capture microdissection of Sample008. (411 KB)

    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.

  7. Supplementary Figure 7: GSEA analysis of genes upregulated in fibroadenoma and UL. (232 KB)

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

PDF files

  1. Supplementary Text and Figures (19,441 KB)

    Supplementary Figures 1-7 and Supplementary Tables 2, 5 and 7

Excel files

  1. Supplementary Table 1 (38.5 KB)

    Clinical characteristics of fibroadenoma patients.

  2. Supplementary Table 3 (35.5 KB)

    List of confirmed somatic mutations identified from whole-exome sequencing of eight fibroadenomas.

  3. Supplementary Table 4 (36 KB)

    Mutations detected in ultra-deep targeted amplicon sequencing of MED12 exon 2 in 98 fibroadenoma samples.

  4. Supplementary Table 6 (42 KB)

    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

  5. Supplementary Table 8 (38)

    Differentially expressed genes between mutant and wild-type MED12 fibroadenoma samples.

Additional data