RNase IIIb Mutations Research Articles

DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer.

DICER1 is a highly conserved RNase III endoribonuclease essential for the biogenesis of single-stranded mature microRNAs (miRNAs) from stem-loop precursor miRNAs. Somatic mutations in the RNase IIIb domain of DICER1 impair its ability to generate mature 5p miRNAs and are believed to drive tumorigenesis in DICER1 syndrome-associated and sporadic thyroid tumors. However, the DICER1-driven specific changes in miRNAs and resulting changes in gene expression are poorly understood in thyroid tissue. In this study, we profiled the miRNA (n=2,083) and mRNA (n=2,559) transcriptomes of 20 non-neoplastic, 8 adenomatous and 60 pediatric thyroid cancers (13 follicular thyroid cancers [FTC] and 47 papillary thyroid cancers [PTC]) of which 8 had DICER1 RNase IIIb mutations. All DICER1-mutant differentiated thyroid cancers (DTC) were follicular patterned (six follicular variant PTC and two FTC), none had lymph node metastasis. We demonstrate that DICER1 pathogenic somatic mutations were associated with a global reduction of 5p-derived miRNAs, including those particularly abundant in the non-neoplastic thyroid tissue such as let-7 and mir-30 families, known for their tumor suppressor function. There was also an unexpected increase of 3p miRNAs, possibly associated with DICER1 mRNA expression increase in tumors harboring RNase IIIb mutations. These abnormally expressed 3p miRNAs, which are otherwise low or absent in DICER1-wt DTC and non-neoplastic thyroid tissues, make up exceptional markers for malignant thyroid tumors harboring DICER1 RNase IIIb mutations. The extensive disarray in the miRNA transcriptome results in gene expression changes, which were indicative of positive regulation of cell-cycle. Moreover, differentially expressed genes point to increased MAPK signaling output and loss of thyroid differentiation comparable to the RAS-like subgroup of PTC (as coined by The Cancer Genome Atlas), which is reflective of the more indolent clinical behavior of these tumors.

Hotspot Mutations in DICER1 Causing GLOW Syndrome-Associated Macrocephaly via Modulation of Specific microRNA Populations Result in the Activation of PI3K/ATK/mTOR Signaling.

We have previously described mosaic mutations in the RNase IIIb domain of DICER1that display global developmental delays, lung cysts, somatic overgrowth, macrocephaly and Wilms tumor. This constellation of phenotypes was classified as GLOW syndrome. Due to the phenotypic overlap between GLOW and syndromes caused by mutations in the PI3K/AKT/mTOR pathway, we hypothesized that alterations in miRNA regulation of this pathway cause its specific constellation of phenotypes. To test the hypothesis that DICER1 "hot spot" mutations associated with GLOW syndrome activate PI3K/AKT/mTOR signaling. We developed HEK293T cells with loss of exon 25 in DICER1, a genetic modification that is synonymous with the "hot spot" RNAseIIIb mutations that cause GLOW syndrome. We assayed the cells for activation of the PI3K/AKT/mTOR signaling pathway. We observed activation of the PI3K/AKT/mTOR pathway as demonstrated by increased pS6Kinase, p4EBP1 and pTSC2 levels. Additionally, these cells demonstrate a striking cellular phenotype, with the ability to form spheres when the serum is removed from their growth medium. The cells in these spheres are Oct4 and Sox2 positive and exhibit the property of reversion with the addition of serum. We queried miRNA expression data and identified a population of miRNAs that increase due to these mutations and target negative regulators of the PI3K/AKT/mTOR pathway. This work identifies the delicate and essential role for miRNA control of the PI3K/AKT/mTOR pathway. We conclude that the phenotypes observed in the GLOW syndrome are the result of PI3K/AKT/mTOR activation.

Deep Sequencing Reveals Spatially Distributed Distinct Hot Spot Mutations in DICER1-Related Multinodular Goiter.

Nontoxic multinodular goiter (MNG) occurs frequently, but its genetic etiology is not well established. Familial MNG and MNG occurring with ovarian Sertoli-Leydig cell tumor are associated with germline DICER1 mutations. We recently identified second somatic DICER1 ribonuclease (RNase) IIIb mutations in two MNGs. The objective of the study was to investigate the occurrence of somatic DICER1 mutations and mutational clonality in MNG. MNGs from 15 patients (10 with and five without germline DICER1 mutations) were selected based on tissue availability. Core biopsies/scrapings (n = 70) were obtained, sampling areas of follicular hyperplasia, hyperplasia within colloid pools, unremarkable thyroid parenchyma, and areas of thyroid parenchyma, not classified. After capture with a Fluidigm access array, the coding sequence of DICER1 was deep sequenced using DNA from each core/scraping. All germline DICER1-mutated cases were found to harbor at least one RNase III mutation. Specifically, we identified 12 individually distinct DICER1 RNase IIIb hot spot mutations in 32 of the follicular hyperplasia or hyperplasia within colloid pools cores/scrapings. These mutations are predicted to affect the metal-ion binding residues at positions p.Glu1705, p.Asp1709, p.Gly1809, p.Asp1810, and p.Glu1813. Somatic RNase IIIb mutations were identified in the 10 DICER1 germline mutated MNGs as follows: two cases contained one somatic mutation, five cases contained two mutations, and three cases contained three distinct somatic hot spot mutations. No RNase IIIb mutations were identified in the MNGs from individuals without germline DICER1 mutations. This study demonstrates that nodules within MNG occurring in DICER1 syndrome are associated with spatially distributed somatic DICER1 RNase IIIb mutations.

Macrocephaly associated with the DICER1 syndrome.

BackgroundGermline mutations in DICER1 increase the risk of various tumors, including pleuropulmonary blastoma. Macrocephaly and symmetric overgrowth has been reported in some, but not all, patients with mosaic DICER1 RNase IIIb mutations; the prevalence of these features in individuals with constitutional germline DICER1 mutations is unknown.MethodsWe analyzed prospectively collected auxology data from 67 DICER1 mutation carriers and 43 family controls. We assessed differences between groups using an exact test for proportions and generalized estimating equations for continuous dependent variables.ResultsTwenty-eight DICER1 mutation carriers (42%) were macrocephalic, and none had an occipital-frontal circumference (OFC) below the 3rd centile, which significantly differed from family controls, of whom five were macrocephalic (12%) and two had OFC below the 3rd centile (5%) (P<0.001). DICER1 mutation carriers were taller than familial controls after controlling for gender (P=0.048), but similar proportions of both groups were above the 97th centile of population norms. Head circumference remained increased after adjusting for differences in height.ConclusionsFor the first time, we establish macrocephaly as a common finding in the DICER1 syndrome. Like some of the other tumor-predisposition disorders, macrocephaly may be a useful, albeit a subtle, clinical clue to the DICER1 syndrome diagnosis.

Abstract 2390: The polyclonal nature of multinodular goitre revealed by DICER1 sequencing

Abstract Nontoxic multinodular goitre (MNG) occurs frequently in the general population and is characterised by nodular enlargement of the thyroid gland. In 2011, Rio Frio et al discovered that familial MNG and MNG occurring with Sertoli-Leydig cell tumours were associated with germ-line mutations in the microRNA-processing gene, DICER1. We recently reported the first instance of biallelic DICER1 mutations in an MNG. This prompted us to further investigate the role of DICER1 mutations in MNG pathogenesis. Given that MNG may either be a monoclonal or polyclonal disease, we set out to investigate whether the molecular landscape was correspondingly heterogeneous. We obtained core biopsies (n = 27) from 9 MNGs. 1mm cores were excised from morphologically distinct regions, including from areas of follicular hyperplasia (FH), hyperplasia within colloid pools (HC) and/or otherwise unremarkable areas. DNA extracted from each core was processed using a custom Fluidigm Access Array followed by deep sequencing. We focused our investigation on the RNase IIIb domain of DICER1 to identify known hotspot mutations. All mutations were validated by Sanger sequencing. We identified 6 individually distinct DICER1 RNase IIIb hotspot mutations in 14 (of 27) cores consisting of FH or HC (Table 1). These mutations affected the metal-ion binding residues at positions 1705, 1709, 1810 &amp; 1813. Two MNGs that arose in individuals without germ-line DICER1 mutations did not carry any RNase IIIb hits. Of the remaining 7 germ-line DICER1-mutated cases, 2 carried 1 RNase IIIb mutation; 4 carried 2 different RNase IIIb mutations and one case did not carry any successfully-validated RNase IIIb mutations. We demonstrate that spatial heterogeneity of RNase IIIb DICER1 mutations exists in MNG. We postulate that cyclic hyperplasia followed by regression over time in response to external stimuli, particularly in germ-line DICER1 mutation carriers, contributes to the polyclonal accumulation of RNase IIIb mutations in MNG. Table 1:DICER1 RNase IIIb Mutation FrequenciesCaseGerm-line DICER1 StatusMorphologyc.5113G&amp;gt;A p.E1705Kc.5125G&amp;gt;A p.D1709Nc.5126A&amp;gt;G p.D1709Gc.5428G&amp;gt;T p.D1810Yc.5429A&amp;gt;T p.D1810Vc.5437G&amp;gt;C p.E1813Q1PositiveHC41.7–––––FH–––––22.12PositiveFH––––––UR––––––HC––––––UR––––––HC––Sanger–––HC––Sanger–––3PositiveHC––––29.2–FH–43.7––––FH–28.8––––4PositiveHC––30.6–––UR––––––HC––33.1–––FH–––52.3––5PositiveHC––––––HC with FH––––––6PositiveFH–––––39.3FH–––––13.1FH––––––7PositiveFH–––41.8––FH28.0–––––8NegativeFH––––––HC––––––9NegativeFH––––––PTC––––––FH––––––Abbreviations: FH, follicular hyperplasia; HC, hyperplasia within colloid pools; UR, unremarkable; PTC, papillary thyroid ca. Notes: Each row corresponds to a separate core. Mutant allele frequencies are indicated in each cell (all &amp;gt;10%); ‘‘–’’ indicates mutation not observed in core DNA; Sanger, indicates mutations previously identified in an independent PCR from bulk MNG DNA, tested by Sanger sequencing. All Fluidigm positives were confirmed by Sanger sequencing from the same DNA that was used to do the Fluidigm analysis. Citation Format: Leanne de Kock, Ismaël Bah, Mona Wu, John R. Priest, Jiannis Ragoussis, William D. Foulkes. The polyclonal nature of multinodular goitre revealed by DICER1 sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2390.

Abstract IA12: Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. Wilms tumor is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, patients suffer adverse late effects of therapy and survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and β-catenin) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor driver mutations in WT1 and β-catenin. DROSHA and DICER1 somatic hotspot mutations occur at or near critical metal-binding residues in RNase III domains and partially or completely disrupt enzyme activity. Examination of miRNA expression in tumors, in vitro processing assays, and genomic editing in human cells demonstrate that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. While DICER1 RNase IIIB mutations preferentially impair processing of miRNAs that derive from the 5′ arm of pre-miRNA hairpins, DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Despite their distinct mechanisms of action, DROSHA and DICER1 mutations result in decreased expression of several common tumor-suppressing miRNAs, including multiple members of the let-7 and miR-15/16 families. We also identified a patient carrying a germline DICER1 loss-of-function allele who subsequently developed multiple malignancies with distinct DICER1 RNase IIIB somatic hotspot mutations, suggesting that DICER1 can act as both a tumor-suppressor and an oncogene depending on the nature of the mutation. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogram miRNA expression in human cancer and suggest that mutations causing impairment of miRNA processing define a distinct subclass of Wilms tumors. Citation Format: Kenneth S. Chen, Dinesh Rakheja, Yangjian Liu, Abhay A. Shukla, Joshua T. Mendell, James F. Amatruda. Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA12.

Abstract 4934: Mosaic RNase IIIb domain DICER1 mutations in children with multiple primary tumors

Abstract The DICER1 syndrome or pleuropulmonary blastoma (PPB) familial tumor and dysplasia syndrome (PPB FTDS) (OMIM #601200) is caused by heterozygous germ-line mutations in the microRNA maturation pathway gene, DICER1. Several rare phenotypes constitute the syndrome including PPB, cystic nephroma (CN), ovarian Sertoli-Leydig cell tumors (SLCT), intra-ocular medulloepithelioma, nasal chondromesenchymal hamartoma (NCMH), pineoblastoma, pituitary blastoma, multinodular goitre (MNG) and other rare childhood sarcomas and dysplasias. Highly characteristic second somatic mutations have been identified in DICER1-associated tumors, affecting amino acid residues central to the catalytic activity of the RNase IIIb domain. We describe four children with multiple primary tumours associated with the DICER1 syndrome. Sanger sequencing of constitutional DNA obtained from peripheral blood lymphocytes and/or saliva revealed no likely deleterious germ-line DICER1 mutations. We subsequently sequenced the region encoding the DICER1 RNase IIIa and RNase IIIb domains in gDNA extracted from the tumor samples, and noted the presence of the same RNase IIIb missense mutation in multiple tumors from each patient (Patient A: c.5437G&amp;gt;C; Patient B: c.5125G&amp;gt;A; Patient C: c.5439G&amp;gt;C; Patient D: c.5425G&amp;gt;A). We performed targeted capture followed by deep sequencing on DNA extracted from both normal and tumor tissue, which revealed the presence of the respective RNase IIIb mutations in a low percentage of sequencing reads (0.2 - 13%) in constitutional DNA from three of the four patients (Patients A, B and C). The relative abundance of the allele harboring the DICER1 RNase IIIb mutation was significantly higher in the tumors compared to normal tissue from the surrounding organ and/or distant sites. Taken together, these findings indicate a mosaic origin of the DICER1 RNase IIIb missense mutations. The mosaic origin of Patient D's mutation remains to be unequivocally established. We further hypothesized that, in the setting of a mosaic DICER1 RNase IIIb mutations, we might discover second somatic mutations outside of the RNase IIIb domain which initiate two-hit tumorigenesis as seen in most DICER1-related tumors. Sequencing data identified individually distinct second somatic, likely-deleterious DICER1 mutations in Patient A's left ovary SLCT and sinonasal inflammatory polyp, in Patient C's NCMH, and in Patient D's Type II PPB which arose in a pre-existing lung cyst. Each of these second somatic mutations are predicted to prematurely truncate the DICER1 protein. We demonstrate that mosaic DICER1 RNase IIIb missense mutations are an occasional and important genetic cause of the DICER1 syndrome in patients presenting with multiple primary tumors associated with the syndrome, and that for tumor initiation, they appear to be accompanied by second somatic truncating non-RNase IIIb DICER1 mutations. Citation Format: Leanne de Kock, Barbara Rivera Polo, Mona Wu, Evan Weber, Claudio Sandoval, Saskia M. J. Hopman, J. Hans M. Merks, Annet van Hagen, D. A. Plager, Nelly Sabbaghian, Nancy Hamel, Dorothée Bouron-Dal Soglio, John R. Priest, William D. Foulkes. Mosaic RNase IIIb domain DICER1 mutations in children with multiple primary tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2015-4934

Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours.

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5′-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.

Germ-line and somatic DICER1 mutations in pineoblastoma.

Germ-line RB-1 mutations predispose to pineoblastoma (PinB), but other predisposing genetic factors are not well established. We recently identified a germ-line DICER1 mutation in a child with a PinB. This was accompanied by loss of heterozygosity (LOH) of the wild-type allele within the tumour. We set out to establish the prevalence of DICER1 mutations in an opportunistically ascertained series of PinBs. Twenty-one PinB cases were studied: Eighteen cases had not undergone previous testing for DICER1 mutations; three patients were known carriers of germ-line DICER1 mutations. The eighteen PinBs were sequenced by Sanger and/or Fluidigm-based next-generation sequencing to identify DICER1 mutations in blood gDNA and/or tumour gDNA. Testing for somatic DICER1 mutations was also conducted on one case with a known germ-line DICER1 mutation. From the eighteen PinBs, we identified four deleterious DICER1 mutations, three of which were germ line in origin, and one for which a germ line versus somatic origin could not be determined; in all four, the second allele was also inactivated leading to complete loss of DICER1 protein. No somatic DICER1 RNase IIIb mutations were identified. One PinB arising in a germ-line DICER1 mutation carrier was found to have LOH. This study suggests that germ-line DICER1 mutations make a clinically significant contribution to PinB, establishing DICER1 as an important susceptibility gene for PinB and demonstrates PinB to be a manifestation of a germ-line DICER1 mutation. The means by which the second allele is inactivated may differ from other DICER1-related tumours.