DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis
Barbara Rivera, … , Marc R. Fabian, William D. Foulkes
Barbara Rivera, … , Marc R. Fabian, William D. Foulkes
Published March 2, 2020; First published December 5, 2019
Citation Information: J Clin Invest. 2020;130(3):1479-1490. https://doi.org/10.1172/JCI130206.
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Clinical Medicine Genetics Oncology

DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis

Abstract

BACKGROUND DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist.METHODS Whole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K–positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K.RESULTS We identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons.CONCLUSION We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDING Canadian Institutes of Health Research, Compute Canada, Alex’s Lemonade Stand Foundation, the Mia Neri Foundation for Childhood Cancer, Cassa di Sovvenzioni e Risparmio fra il Personale della Banca d’Italia, and the KinderKrebsInitiative Buchholz/Holm-Seppensen.

Authors

Barbara Rivera, Javad Nadaf, Somayyeh Fahiminiya, Maria Apellaniz-Ruiz, Avi Saskin, Anne-Sophie Chong, Sahil Sharma, Rabea Wagener, Timothée Revil, Vincenzo Condello, Zineb Harra, Nancy Hamel, Nelly Sabbaghian, Karl Muchantef, Christian Thomas, Leanne de Kock, Marie-Noëlle Hébert-Blouin, Angelia V. Bassenden, Hannah Rabenstein, Ozgur Mete, Ralf Paschke, Marc P. Pusztaszeri, Werner Paulus, Albert Berghuis, Jiannis Ragoussis, Yuri E. Nikiforov, Reiner Siebert, Steffen Albrecht, Robert Turcotte, Martin Hasselblatt, Marc R. Fabian, William D. Foulkes

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Figure 4

Differentially expressed mRNA and miRNA analysis of tumors with or without DGCR8 mutation and in vitro cleavage of miR-30c-2.

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Differentially expressed mRNA and miRNA analysis of tumors with or witho...
For all 3 volcano plots, log fold-change is plotted on the x axis and the adjusted P value (FDR; –log10 scale) on the y axis. Dotted horizontal and vertical lines indicate threshold of significance (FDR < 0.01) and absolute fold change (>2). Over- and underexpressed mRNAs/miRNAs in mutated cases compared with WT are shown in red and blue, respectively. Black dots represent the mirtrons. (A) Results of differentially expressed mRNA expression analysis between schwannomas with (n = 4) and without (n = 5) the c.1552G>A;p.E518K mutation. (B) Results of differentially expressed miRNA expression analysis between schwannomas with (n = 4) and without (n = 5) the c.1552G>A;p.E518K mutation. One of the most significantly underexpressed pre-miRNAs (as shown in Supplemental Figure 12), miR-30c-2, is a precursor to the most significantly underexpressed mature miRNA. All significant mirtrons are overrepresented in mutated cases compared with WT. The top 25 mRNAs and miRNAs, up and down, are listed in Supplemental Tables 9 and 10, respectively. (C) In vitro cleavage results of pri-miR30c-2. The microprocessor complex formed with DGCR8-WT trims the primary miRNA into a precursor miRNA, but the mutant DGCR8-E518K fails to cleave the primary miRNA even after 60 minutes. Reaction incubation time is shown in minutes. The image is representative of 3 independent replicate experiments. In each experiment, 3 conditions were tested, and freshly immunoprecipitated proteins were used in each case. DGCR8-WT and DROSHA, pull down of the complex between DGCR8-WT protein and endogenous DROSHA; DGCR8-E518K and DROSHA, pull down of the complex between DGCR8-E518K mutant protein and endogenous DROSHA; M, RNA-labeled ladder marker; black arrow shows the band corresponding to the primary miRNA (approximately 100 bp); gray arrow points the band corresponding to a precursor miRNA (approximately 65 bp). (D) Differentially expressed miRNA expression analysis between Wilms tumors with (n = 4) and without (n = 20) DGCR8-E518K mutation. All significant mirtrons are overrepresented compared with WT tumors.