Recurrent EZH1 mutations are a second hit in autonomous thyroid adenomas
Davide Calebiro, … , Luca Persani, Ralf Paschke
Davide Calebiro, … , Luca Persani, Ralf Paschke
Published August 8, 2016
Citation Information: J Clin Invest. 2016;126(9):3383-3388. https://doi.org/10.1172/JCI84894.
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Concise Communication Endocrinology

Recurrent EZH1 mutations are a second hit in autonomous thyroid adenomas

Abstract

Autonomous thyroid adenomas (ATAs) are a frequent cause of hyperthyroidism. Mutations in the genes encoding the TSH receptor (TSHR) or the Gs protein α subunit (GNAS) are found in approximately 70% of ATAs. The involvement of other genes and the pathogenesis of the remaining cases are presently unknown. Here, we performed whole-exome sequencing in 19 ATAs that were paired with normal DNA samples and identified a recurrent hot-spot mutation (c.1712A>G; p.Gln571Arg) in the enhancer of zeste homolog 1 (EZH1) gene, which codes for a catalytic subunit of the polycomb complex. Targeted screening in an independent cohort confirmed that this mutation occurs with high frequency (27%) in ATAs. EZH1 mutations were strongly associated with known (TSHR, GNAS) or presumed (adenylate cyclase 9 [ADCY9]) alterations in cAMP pathway genes. Furthermore, functional studies revealed that the p.Gln571Arg EZH1 mutation caused increased histone H3 trimethylation and increased proliferation of thyroid cells. In summary, this study revealed that a hot-spot mutation in EZH1 is the second most frequent genetic alteration in ATAs. The association between EZH1 and TSHR mutations suggests a 2-hit model for the pathogenesis of these tumors, whereby constitutive activation of the cAMP pathway and EZH1 mutations cooperate to induce the hyperproliferation of thyroid cells.

Authors

Davide Calebiro, Elisa S. Grassi, Markus Eszlinger, Cristina L. Ronchi, Amod Godbole, Kerstin Bathon, Fabiana Guizzardi, Tiziana de Filippis, Knut Krohn, Holger Jaeschke, Thomas Schwarzmayr, Rifat Bircan, Hulya Iliksu Gozu, Seda Sancak, Marek Niedziela, Tim M. Strom, Martin Fassnacht, Luca Persani, Ralf Paschke

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Figure 3

Functional characterization of the Gln571Arg EZH1 mutant.

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Functional characterization of the Gln571Arg EZH1 mutant. (A) Effect of ...
(A) Effect of EZH1 Gln571Arg on histone H3 methylation at Lys27. The methylation pattern in lysates from cells transfected with FLAG-tagged EZH1 WT/Gln571Arg or the empty expression vector (control) was evaluated by Western blot analysis with Abs against mono- (H3K27me1), di- (H3K27me2), and trimethylation (H3K27me3). The expression of transfected EZH1 was evaluated using an Ab against the FLAG tag. (B) H3K27 methylation in thyroid cell lines stably (FRT) or transiently (PCCL3) transfected with EZH1 WT/Gln571Arg. (C) H3K27 methylation in ATA samples with or without Gln571Arg EZH1 mutation and the corresponding NSTs. (D) Proliferation of FRT cells stably expressing EZH1 WT/Gln571Arg. Cell viability was measured with the MTT assay. Data (n = 8) are shown as the mean ± SEM. ***P < 0.001 versus control; §§P < 0.01 versus EZH1 WT; §§§P < 0.001 versus EZH1 WT. Statistical significance was determined by 2-way ANOVA, followed by Bonferroni’s post test (E) Western blot analysis of proliferative markers in FRT cells stably expressing EZH1 WT/Gln571Arg. Results in A, B, and E are representative of 3 independent experiments. p-H3, phosphorylated H3.