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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- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Supplementary Text and Figures (19,441 KB)
Supplementary Figures 1-7 and Supplementary Tables 2, 5 and 7
- Supplementary Table 1 (38.5 KB)
Clinical characteristics of fibroadenoma patients.
- Supplementary Table 3 (35.5 KB)
List of confirmed somatic mutations identified from whole-exome sequencing of eight fibroadenomas.
- Supplementary Table 4 (36 KB)
Mutations detected in ultra-deep targeted amplicon sequencing of MED12 exon 2 in 98 fibroadenoma samples.
- 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
- Supplementary Table 8 (38)
Differentially expressed genes between mutant and wild-type MED12 fibroadenoma samples.