Ten years of DICER1 mutations: Provenance,distribution, and associated phenotypes

DICER1 syndrome is a pleiotropic tumor predisposition syndrome characterized by a distinctive constellation of neoplastic and dysplastic lesions, which are generally rare and affect children and young adults. Germline pathogenic variants in the DICER1 gene are the underlying cause of the syndrome; variants are typically inherited in an autosomal dominant pattern but may arise de novo in the germline or in a somatic mosaic distribution. The encoded DICER1 protein is a key component of the microRNA processing pathway. In this Mutation Update, we present a comprehensive review of all DICER1 genetic alterations reported in articles published before January 31st, 2019. A total of 1,136 DICER1 alterations were catalogued from 808 individuals and the tumors that occurred in these persons. We provide an inventory of these DICER1 alterations and discuss them with respect to their provenance, distribution across the gene, associated phenotypes and ages of onset, and penetrance. We also address approaches to classification of DICER1 variants of uncertain significance and discuss the clinical significance of DICER1 variant identification.     

Cell‐ and developmental stage‐specific Dicer1 ablation in the lung epithelium models cystic pleuropulmonary blastoma

Inherited syndromes provide unique opportunities to identify key regulatory mechanisms governing human disease. We previously identified germline loss‐of‐function DICER1 mutations in a human syndrome defined by the childhood lung neoplasm pleuropulmonary blastoma (PPB), which arises during lung development. DICER1 regulates many biological processes critical in development and disease pathogenesis. Significant challenges in defining the role of DICER1 in human disease are identifying cause–effect relationships and generating manipulatable systems that model the complexity of organ development and disease pathogenesis. Here we report the generation of a murine model for PPB and demonstrate that precise temporal and cell type‐specific Dicer1 ablation is necessary and sufficient for the development of cystic lungs that histologically and phenotypically model PPB. Dicer1 ablation in the distal airway epithelium during early stages of lung development resulted in a cystic lung phenotype indistinguishable from PPB, whereas DICER1 function was not required for development of the proximal airway epithelium or during later stages of organogenesis. Mechanistic studies demonstrate that Dicer1 loss results in epithelial cell death, followed by cystic airway dilatation accompanied by epithelial and mesenchymal proliferation. These studies define precise temporal and epithelial cell type‐specific DICER1 functions in the developing lung and demonstrate that loss of these DICER1 functions is sufficient for the development of cystic PPB. These results also provide evidence that PPB arise through a novel mechanism of non‐cell‐autonomous tumour initiation, in which the genetic abnormality initiating the neoplasm does not occur in the cells that ultimately transform, but rather occurs in a benign‐appearing epithelial cell component that predisposes underlying mesenchymal cells to malignant transformation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An autopsy case of prostatic rhabdomyosarcoma with DICER1 hotspot mutation

Somatic hotspot DICER1 mutations, which frequently coexist with germline inactivating mutation (i.e., DICER1 syndrome), have been identified in various types of benign and malignant conditions. Herein, we report an autopsy case of prostatic rhabdomyosarcoma (RMS) with a hotspot DICER1 c.5125G>A (p.D1709N) mutation. A 26 year-old man presented with a prostatic mass, hematuria, and urinary retention. He underwent total pelvic exenteration, colostomy, ileal conduit construction and partial urethrectomy. Five months postoperatively, he developed multiple metastases to the lungs, brain, iliopsoas muscles and bones. He died of respiratory failure, and autopsy was performed. Microscopically, the tumor was primarily composed of uniform primitive mesenchymal cells infiltrating to the prostate with cambium layer. Rhabdomyoblasts and anaplastic cells were focally observed. Immunohistochemically, tumor cells were positive for desmin, myogenin, PAX7, HMGA2. Multinodular goiter was detected at autopsy. Because the morphology is similar to pleuropulmonary blastoma and DICER1-mutant RMS of the female genital tract, we tested and identified a hotspot DICER1 mutation with Sanger sequencing. Recognizing DICER1-mutant tumor is important because of its frequent association with germline DICER1 inactivation and potential therapeutic implication. Further research is needed to clarify whether this case can be classified as embryonal RMS with anaplasia or ‘DICER1-associated sarcoma’.     

Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis

DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic 'hotspot' mutations at the four metal‐binding sites within the RNase IIIb domain of DICER1 were identified in ovarian sex cord‐stromal tumours and have since been described in other paediatric tumours. In this study, we screened the RNase IIIb domain of DICER1 in 290 endometrial tumours and identified six cases with hotspot mutations, including two cases affected by an atypical G1809R mutation directly adjacent to a metal‐binding site. Using Illumina and Sanger targeted resequencing, we observed and validated biallelic DICER1 mutations in several cases with hotspot mutations. Through in vitro DICER1 cleavage assays, small RNA deep sequencing and real‐time PCR, we demonstrated that mutations adding a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production to mutations at the metal‐binding sites. As expected, 5p miRNAs were globally reduced in tumours and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes de‐repressed due to loss of 5p miRNAs are strongly associated with pathways regulating the cell cycle. Using a Dicer1‐null mouse cell line model, we found that expression of DICER1 hotspot mutants promoted cell proliferation, whereas wild‐type (WT) DICER1 inhibited cell proliferation. Furthermore, targets of let‐7 family miRNAs are enriched among the up‐regulated genes, suggesting that loss of let‐7 may be impacting downstream pathways. Our results reveal that DICER1 hotspot mutations are implicated in common malignancies and may constitute a unique oncogenic pathway. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Multiple DICER1‐related tumors in a child with a large interstitial 14q32 deletion

Germ‐line interstitial deletions involving the 14q32 chromosomal region, resulting in 14q32 deletion syndrome, are rare. DICER1 is a recently described cancer‐predisposition gene located at 14q32.13. We report the case of a male child with a ～5.8 Mbp 14q32.13q32.2 germ‐line deletion, which included the full DICER1 locus. We reviewed available clinical and pathological material, and conducted genetic analyses. In addition to having congenital dysmorphic features, the child developed multiple DICER1 syndrome‐related tumors before age 5 y: a pediatric cystic nephroma (pCN), a ciliary body medulloepithelioma (CBME), and a small lung cyst (consistent with occult pleuropulmonary blastoma Type I/Ir cysts seen in DICER1 mutation carriers). He also developed a cerebral spindle‐cell sarcoma with myogenous differentiation. Our investigations revealed that the deletion encompassed 31 protein‐coding genes. In addition to the germ‐line DICER1 deletion, somatic DICER1 RNase IIIb mutations were found in the CBME (c.5437G > A, p.E1813K), pCN (c.5425G > A, p.G1809R), and sarcoma (c.5125G > A, p.D1709N). The sarcoma also harbored a somatic TP53 mutation: c.844C > T, p.R282W. Additional copy number alterations were identified in the CBME and sarcoma using an OncoScan array. Among the 8 cases with molecularly‐defined 14q32 deletions involving DICER1 and for whom phenotypic information is available, our patient and one other developed DICER1‐related tumors. Biallelic DICER1 mutations have not previously been reported to cause cerebral sarcoma, which now may be considered a rare manifestation of the DICER1 syndrome. Our study shows that DICER1‐related tumors can occur in children with 14q32 deletions and suggests surveillance for such tumors may be warranted.     

Germline mutations and new copy number variants among 40 pediatric cancer patients suspected for genetic predisposition

Cancer predisposition syndromes (CPS) result from germline pathogenic variants, and they are increasingly recognized in the etiology of many pediatric cancers. Herein, we report the genetic/genomic analysis of 40 pediatric patients enrolled from 2016 to 2018. Our diagnostic workflow was successful in 50% of screened cases. Overall, the proportion of CPS in our case series is 10.9% (20/184) of enrolled patients. Interestingly, 12.5% of patients achieved a conclusive diagnosis through the analysis of chromosomal imbalance. Indeed, we observed germline microdeletions/duplications of regions encompassing cancer-related genes in 50% of patients undergoing array-CGH: EIF3H duplication in a patient with infantile desmoplastic astrocytoma and low-grade Glioma; SLFN11 deletion, SOX4 duplication, and PARK2 partial deletion in three neuroblastoma patients; a PTPRD partial deletion in a child diagnosed with glioblastoma multiforme. Finally, we identified two cases due to DICER1 germline mutations.     

Cancer‐associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse‐strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non‐epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal‐binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3′ end of the 5p miRNA strand of a pre‐mRNA hairpin. To investigate the effects of these cancer‐associated 'hotspot' mutations, we engineered mouse DICER1‐deficient ES cells to express wild‐type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal‐binding site mutations were compared to each other and to wild‐type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation‐carrying cells were distinct from both wild‐type and DICER1‐deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p‐derived miRNAs. We therefore conclude that cancer‐associated somatic hotspot mutations of DICER1, affecting any one of four metal‐binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Methanogenic archaea in subgingival sites: a review

Archaea are non‐bacterial prokaryotes associated with oral microbiota in humans, but their roles in oral pathologies remain controversial. Several studies reported the molecular detection of methanogenic archaea from periodontitis, but the significance of this association has not been confirmed yet. An electronic search was therefore conducted in MEDLINE‐Pubmed to identify all papers published in English connecting archaea and periodontal infections. Data analysis of the selected studies showed that five genera of methanogenic archaea have been detected in the subgingival microbiota, Methanobrevibacter oralis being the most frequently detected species in 41% of periodontitis patients and 55% of periodontal pockets compared to 6% of healthy subjects and 5% of periodontally‐healthy sites (p < 10^?5, Chi‐squared test). Based on the five determination‐criteria proposed by Socransky (association with disease, elimination of the organism, host response, animal pathogenicity and mechanisms of pathogenicity), M. oralis is a periodontal pathogen. The methanogenic archaea load correlating with periodontitis severity further supports the pathogenic role of methanogenic archaea in periodontitis. Therefore, detection and quantification of M. oralis in periodontal pockets could help the laboratory diagnosis and follow‐up of periodontitis. Determining the origin, diversity and pathogenesis of archaea in periodontal infections warrants further investigations.     

Identification of two 14q32 deletions involving DICER1 associated with the development of DICER1-related tumors

DICER1 encodes an RNase III endonuclease protein that regulates the production of small non-coding RNAs. Germline mutations in DICER1 are associated with an autosomal dominant hereditary cancer predisposition syndrome that confers an increased risk for the development of several rare childhood and adult-onset tumors, the most frequent of which include pleuropulmonary blastoma, ovarian sex cord-stromal tumors, cystic nephroma, and thyroid gland neoplasia. The majority of reported germline DICER1 mutations are truncating sequence-level alterations, suggesting that a loss-of-function type mechanism drives tumor formation in DICER1 syndrome. However, reports of patients with germline DICER1 whole gene deletions are limited, and thus far, only two have reported an association with tumor development. Here we report the clinical findings of three patients from two unrelated families with 14q32 deletions that encompass the DICER1 locus. The deletion identified in Family I is 1.4?Mb and was initially identified in a 6-year-old male referred for developmental delay, hypotonia, macrocephaly, obesity, and behavioral problems. Subsequent testing revealed that this deletion was inherited from his mother, who had a clinical history that included bilateral multinodular goiter and papillary thyroid carcinoma. The second deletion is 5.0?Mb and was identified in a 15-year-old female who presented with autism, coarse facial features, Sertoli-Leydig cell tumor, and Wilms’ tumor. These findings provide additional supportive evidence that germline deletion of DICER1 confers an increased risk for DICER1-related tumor development, and provide new insight into the clinical significance of deletions involving the 14q32 region.     

PMS2 involvement in patients suspected of Lynch syndrome

It is well‐established that germline mutations in the mismatch repair genes MLH1, MSH2, and MSH6 cause Lynch syndrome. However, mutations in these three genes do not account for all Lynch syndrome (suspected) families. Recently, it was shown that germline mutations in another mismatch repair gene, PMS2, play a far more important role in Lynch syndrome than initially thought. To explore this further, we determined the prevalence of pathogenic germline PMS2 mutations in a series of Lynch syndrome‐suspected patients. Ninety‐seven patients who had early‐onset microsatellite instable colorectal or endometrial cancer, or multiple Lynch syndrome‐associated tumors and/or were from an Amsterdam Criteria II‐positive family were selected for this study. These patients carried no pathogenic germline mutation in MLH1, MSH2, or MSH6. When available, tumors were investigated for immunohistochemical staining (IHC) for PMS2. PMS2 was screened in all patients by exon‐by‐exon sequencing. We identified four patients with a pathogenic PMS2 mutation (4%) among the 97 patients we selected. IHC of PMS2 was informative in one of the mutation carriers, and in this case, the tumor showed loss of PMS2 expression. In conclusion, our study confirms the finding of previous studies that PMS2 is more frequently involved in Lynch syndrome than originally expected. © 2009 Wiley‐Liss, Inc.     

Germline large deletion of BAP1 and decreased expression in non‐tumor choroid in uveal melanoma patients with high risk for inherited cancer

Uveal melanoma (UM) is the most common phenotype in patients with germline BAP1 mutation. This study aimed to identify selection criteria for BAP1 germline testing and assessed the role of large deletion/duplication and epigenetic inactivation. One hundred seventy‐two UM patients with high risk of hereditary cancer were included. Germline variants in BAP1 were assessed by direct sequencing and large deletion/duplication by multiplex ligation‐dependent probe amplification. BAP1 expression in unaffected choroid tissue from a patient with UM was assessed by quantitative RT‐PCR and methylation by pyrosequencing. Twenty‐eight patients had one or more germline sequence variants in BAP1; seven of these were pathogenic. One hundred forty patients were assessed for large deletion/duplication and in one BAP1 whole gene deletion was detected. In total, eight patients (4.7%) had pathogenic alterations in BAP1 with the highest frequencies of in patients with a personal/family history of ≥2 BAP1‐related cancers 6/16 (38%), age of onset <35 years 4/21 (19%) and familial UM 6/34 (18%). One of 19 non‐tumor choroid tissues tested showed uncharacteristically low expression as compared to the controls decrease in BAP1 RNA expression but no evidence of constitutional promotor hypermethylation was detected. UM patients with a strong personal or family history of cancers associated with BAP1, early age of onset and familial UM should be assessed for germline variants in BAP1, including large deletions.     

Comprehensive profiling of BRCA1 and BRCA2 variants in breast and ovarian cancer in Chinese patients

The identification and interpretation of germline BRCA1/2 variants become increasingly important in breast and ovarian cancer (OC) treatment. However, there is no comprehensive analysis of the germline BRCA1/2 variants in a Chinese population. Here we performed a systematic review and meta‐analysis on such variants from 94 publications. A total of 2,128 BRCA1/2 variant records were extracted, including 601 from BRCA1 and 632 from BRCA2. In addition, 414, 734, 449, and 307 variants were also recorded in the BIC, ClinVar, ENIGMA, and UMD databases, respectively, and 579 variants were newly reported. Subsequent analysis showed that the overall germline BRCA1/2 pathogenic variant frequency was 5.7% and 21.8% in Chinese breast and OC, respectively. Populations with high‐risk factors exhibited a higher pathogenic variant percentage. Furthermore, the variant profile in Chinese is distinct from that in other ethnic groups with no distinct founder pathogenic variants. We also tested our in‐house American College of Medical Genetics‐guided pathogenicity interpretation procedure for Chinese BRCA1/2 variants. Our results achieved a consistency of 91.2–97.6% (5‐grade classification) or 98.4–100% (2‐grade classification) with public databases. In conclusion, this study represents the first comprehensive meta‐analysis of Chinese BRCA1/2 variants and validates our in‐house pathogenicity interpretation procedure, thereby providing guidance for further PARP inhibitor development and companion diagnostics in the Chinese population.     

Mutations in the <Emphasis Type="BoldItalic">ELANE</Emphasis> Gene are Associated with Development of Periodontitis in Patients with Severe Congenital Neutropenia

Background  

Patients with severe congenital neutropenia (SCN) often develop periodontitis despite standard medical and dental care. In light of previous findings that mutations in the neutrophil elastase gene, ELANE, are associated with more severe neutropenic phenotypes, we hypothesized an association between the genotype of SCN and development of periodontitis.     

A review of DICER1: structure,function and contribution to disease

DICER1 is an important housekeeping gene that encodes a multi-domain enzyme that functions in small RNA processing. The enzymatic products function in several pathways including RNA interference, DNA damage repair and antiviral response. At the protein level, endoribonuclease DICER also plays a role in many human illnesses, and much research is being done to clarify the full spectrum of its functions and relation to neoplastic and non-neoplastic disease. Both somatic and germline mutations in DICER1 have been identified, and germline mutations have been associated with at least two named syndromes: DICER1 syndrome and GLOW syndrome. The ubiquitous nature of this enzyme makes it an attractive target for future study and therapeutic potential, but much remains to be learned.     

Variable population prevalence estimates of germline TP53 variants: A gnomAD‐based analysis

Reports of variable cancer penetrance in Li–Fraumeni syndrome (LFS) have raised questions regarding the prevalence of pathogenic germline TP53 variants. We previously reported higher‐than‐expected population prevalence estimates in sequencing databases composed of individuals unselected for cancer history. This study aimed to expand and further evaluate the prevalence of pathogenic and likely pathogenic germline TP53 variants in the gnomAD dataset (version r2.0.2, n = 138,632). Variants were selected and classified based on our previously published algorithm and compared with alternative estimates based on three different classification databases: ClinVar, HGMD, and the UMD_TP53 database. Conservative prevalence estimates of pathogenic and likely pathogenic TP53 variants were within the range of one carrier in 3,555–5,476 individuals. Less stringent classification increased the approximate prevalence to one carrier in every 400–865 individuals, mainly due to the inclusion of the controvertible p.N235S, p.V31I, and p.R290H variants. This study shows a higher‐than‐expected population prevalence of pathogenic and likely pathogenic germline TP53 variants even with the most conservative estimates. However, these estimates may not necessarily reflect the prevalence of the classical LFS phenotype, which is based upon family history of cancer. Comprehensive approaches are needed to better understand the interplay of germline TP53 variant classification, prevalence estimates, cancer penetrance, and LFS‐associated phenotype.     

Prevalence of germline mutations in the spindle assembly checkpoint gene BUB1B in individuals with early‐onset colorectal cancer

Germline mutations in BUB1B, encoding BUBR1, one of the crucial components of the spindle assembly checkpoint (SAC), have been shown to cause variable phenotypes, including the recessive mosaic variegated aneuploidy (MVA) syndrome, which predisposes to cancer. Reduced levels of the wild‐type BUBR1 protein have been linked to the development of gastrointestinal neoplasms. To determine whether mutations in BUB1B are enriched in individuals with colorectal cancer (CRC), we performed amplicon‐based targeted next‐generation sequencing of BUB1B on germline DNA of 192 individuals with early‐onset CRC (≤50 years). None of the individuals was found to be homozygous or compound heterozygous for mutations in BUB1B. However, we did identify two rare heterozygous variants, p.Glu390del and p.Cys945Tyr, in patients who developed CRC at the ages of 41 and 43 years, respectively. Both variants were shown not to affect BUBR1 protein expression levels and protein localization. Since the p.Glu390del variant is located in the BUB3‐binding domain, we also performed immunoprecipitation to examine whether this variant affects the binding of BUB1 or BUB3 to BUBR1 but, compared to wild‐type BUBR1, no difference was observed. Our data suggest that mutations in BUB1B do not occur frequently in the germline of individuals with CRC and that BUB1B unlikely plays a major role in the predisposition to early‐onset CRC. Whether carriers of pathogenic BUB1B mutations, such as the parents of MVA syndrome patients, have an increased risk for cancer remains of interest, as studies in mice have suggested that haploinsufficiency of BUB1B may cause an increase in carcinogen‐induced tumors. © 2016 Wiley Periodicals, Inc.     

Providing more evidence on LZTR1 variants in Noonan syndrome patients

Noonan syndrome (NS, OMIM 163950) is a common autosomal dominant RASopathy caused mainly by gain‐of‐function germline pathogenic variants in genes involved in the RAS/MAPK signaling pathway. LZTR1 gene has been associated with both dominant and recessive NS. Here, we present seven patients with NS and variants in the LZTR1 gene from seven unrelated families, 14 individuals in total. The detection rAte of LZTR1 variants in our NS cohort was 4% similar to RAF1 and KRAS genes, indicating that variants in this gene might be frequent among our population. Three different variants were detected, c.742G>A (p.Gly248Arg), c.360C>A (p.His120Gln), and c.2245T>C (p.Tyr749His). The pathogenic variant c.742G>A (p.Gly248Arg) was found in five/seven patients. In our cohort 50% of patients presented heart defects and neurodevelopment delay or learning disabilities, short stature was present in 21% of them and one patient had acute lymphoblastic leukemia. This study broadens the spectrum of variants in the LZTR1 gene and provides increased knowledge of the clinical phenotypes observed in Argentinean NS patients.