Somatic MED12 Mutations in Myometrial Cells

Abstract

Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.

Keywords: MED12 mutation; duplex deep sequencing; leiomyoma; myometrium.

Figure 1

Point mutations of MED12 exon 2 detected by DDS in 29 cases. (A) A diagram illustrating the myometrial samples used for DDS. (B) Mutation plot showing the distribution of point mutations in exon 2 for 31 cases. Red circle highlights the 2 outliers. (C) Dot plot highlighting 2 outliers in the myometrium of 2 cases with >5 LM. (D) Mutation plot shows exon 2 mutation distribution in 29 cases by excluding 2 outliers. (E) Two-dimensional map illustrating the distribution of MED12 exon 2 mutations in each of 29 cases. Mutation frequency is marked from blue (low) to high (red). (F) Dot plot illustrating the MED12 mutations in c.130-131 and other sites.

Figure 2

Mutation analysis of MED12 exon 2 in association with race, number of tumors and age. (A,B) Mutation rate of MED12 exon 2 (A) and c.130-131 (B) in myometrium of black (n = 16) and white (n = 13) women. (C) Mutation rate of MED12 exon 2 in myometrium with ≥5 and <5 LM. (D,E). Mutation rate of MED12 exon 2 (D) and c.130-c.131 (E) in association with patient’s age. ns: no statistical significance.

Figure 3

MED12 exon 2 mutation signature analysis. (A) Distribution of mutation frequency in each of the 8 mutation signatures in 29 cases. (B,C) Accumulated point mutations and percentage in G, C, A, and T nucleotides. (D) Mutation rate of G > A, C, T in c.130-c.131.

Figure 4

Mutation analysis of TP53 exon 5 in 29 cases. (A) Two-dimensional distribution of base mutations in each of 29 cases. Mutation rate was indicated from low (blue) to high (red) bar. (B) 7 mutation signatures detected in TP53 exon 5. (C,D) Accumulated point mutations and percentage in G, C, A, and T nucleotides in TP53 exon 5.