DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis

Abstract

BACKGROUNDDICER1 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.METHODSWhole-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.RESULTSWe 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.CONCLUSIONWe identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDINGCanadian 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.

Keywords: Genetic diseases; Genetics; Oncology; RNA processing; Thyroid disease.

Figure 1. Pedigree of the family: clinical data and genotypes of a family kindred with germline DGCR8 variant c.1552G>A, p.E518K.

dx, diagnosis. Person I-1 was diagnosed with a multinodular goiter (MNG) and with a schwannoma (schw). Person II-1 was diagnosed with MNG. Person II-2 had MNG, a mature cystic teratoma, 9 schwannomas, and an ovarian serous cystadenofibroma. Individual III-1 was diagnosed with autism spectrum disorder (ASD) and MNG, and had a choroid plexus papilloma (CPP) WHO stage I and multiple schwannomas. III-3 was diagnosed with MNG and multiple schwannomas. Person III-2 was diagnosed with MNG and a single schwannoma located in the right knee. Chromatograms display the c.1552G>A;p.E518K locus in germline DNA (gDNA, blue circle) and tumor/MNG DNA (tissue DNA [tDNA], red circle) for each affected individual. Representative chromatograms show LOH in individuals I-1, II-1, II-2, III-1, III-2, and III-3. Schwannoma samples from III-2 and III-3 had a remnant of the WT allele, likely due to normal tissue contamination. The chromatograms for the MNG tDNA in III-1, III-2, and III-3 are representative of the Sanger sequencing results for all 3 nodules in each patient. All results are summarized in Supplemental Tables 3–6 and Supplemental Figures 5 and 6. Arrow indicates a WT genotype. Asterisk indicates presence of the mutant base. Three nodules of the MNG were sequenced for II-1, III-1, III-2, and III-3. Germline DNA with WT sequence at the c.1552G;p.E518 locus is shown for unaffected individual III-5. No tumor DNA from the schwannomas of III-1 and from the MNG of I-1 was available.

Figure 2. Canonical versus intron-derived miRNA production pathway.

Schematic diagrams of canonical miRNA processing pathway (left) and the intronic miRNA (mirtrons) processing pathway (right). Mirtrons are processed by the spliceosome in a manner completely independent of the microprocessor. Both pre-miRNAs and pre-mirtrons are then exported to the cytoplasm and further processed by DICER1. A close-up representation of the microprocessor in trimeric state (2 molecules of DGCR8 and 1 DROSHA) and the pri-miRNA are shown in the inset.

Figure 3. Clustering of miRNA expression in DGCR8-c.1552G>A;p.E518K mutated tumors and their WT counterparts.

(A) Clustering of miRNA expression in 4 DGCR8-E518K mutated schwannomas, 5 DGCR8-WT schwannomas, 4 DGCR8-E518K mutated, and 20 DGCR8-WT Wilms tumors analyzed by the TARGET initiative (7). Heatmap shows sample-by-sample correlation matrix, based on Pearson correlation coefficients, using normalized values for 300 most variable miRNA expression across all samples. The DGCR8-E518K mutated tumors clustered together in the same consensus cluster. DGCR8 denotes which cases harbor the DGCR8-c.1552G>A;p.E518K variant (mut) and which are DGCR8-WT. See also Supplemental Figures 8 and 9 and ref. . (B) Unsupervised clustering of the top 50 most variably expressed miRNAs based on NanoString data of 8 MNG samples (5 DGCR8-E518K mutated and 3 WT cases), 4 follicular variant papillary thyroid cancers (PTC) (2 DGCR8-E518K mutated and 2 WT), and 11 schwannomas (5 DGCR8-E518K mutated; 1 with the VUS c.1147A>G;p.S383G case [*]; 1 with germline variant c.1763A>G;p.K588R previously described in Wilms tumors [#]; and 4 WT for DGCR8). DGCR8-E518K mutated samples clustered together independently of the tissue of origin. Both c.1147A>G;p.S383G and c.1763A>G;p.K588R variants clustered with WT tumors, suggesting the variants do not disturb the miRNA processing role of DGCR8. While tissue types are distributed randomly among DGCR8-E518K, WT samples clustered by tissue of origin (thyroid vs. schwannoma cells), highlighting the particularity of the c.1552G>A;p.E518K profile in miRNA processing.

Figure 4. Differentially expressed mRNA and miRNA analysis of tumors with or without DGCR8 mutation and in vitro cleavage of miR-30c-2.

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.