Dorothée Bouron-Dal Soglio

DICER1 Mutations in Familial Multinodular Goiter With and Without Ovarian Sertoli-Leydig Cell Tumors

CONTEXT Nontoxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported, and published reports describe 5 families that also contain at least 1 individual with a Sertoli-Leydig cell tumor of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have been identified in families affected by pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCTs. OBJECTIVE To determine whether familial MNG with or without SLCT in the absence of PPB was associated with mutations in DICER1. DESIGN, SETTING, AND PATIENTS From September 2009 to September 2010, we screened 53 individuals from 2 MNG and 3 MNG/SLCT families at McGill University for mutations in DICER1. We investigated blood lymphocytes and MNG and SLCT tissue from family members for loss of the wild-type DICER1 allele (loss of heterozygosity), DICER1 expression, and microRNA (miRNA) dysregulation. MAIN OUTCOME MEASURE Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. RESULTS We identified and characterized germline DICER1 mutations in 37 individuals from 5 families. Two mutations were predicted to be protein truncating, 2 resulted in in-frame deletions, and 1 was a missense mutation. Molecular analysis of the 3 SLCTs showed no loss of heterozygosity of DICER1, and immunohistochemical analysis in 2 samples showed strong expression of DICER1 in Sertoli cells but weak staining of Leydig cells. miRNA profiling of RNA from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. CONCLUSIONS DICER1 mutations are associated with both familial MNG and MNG with SLCT, independent of PPB. These germline DICER1 mutations are associated with dysregulation of miRNA expression patterns.

Extending the phenotypes associated with DICER1 mutations

DICER1 is crucial for embryogenesis and early development. Forty different heterozygous germline DICER1 mutations have been reported worldwide in 42 probands that developed as children or young adults, pleuropulmonary blastoma (PPB), cystic nephroma (CN), ovarian sex cord‐stromal tumors (especially Sertoli‐Leydig cell tumor [SLCT]), and/or multinodular goiter (MNG). We report DICER1 mutations in seven additional families that manifested uterine cervix embryonal rhabdomyosarcoma (cERMS, four cases) and primitive neuroectodermal tumor (cPNET, one case), Wilms tumor (WT, three cases), pulmonary sequestration (PS, one case), and juvenile intestinal polyp (one case). One carrier developed (age 25 years) a pleomorphic sarcoma of the thigh; another carrier had transposition of great arteries (TGA). These observations show that cERMS, cPNET, WT, PS, and juvenile polyps fall within the spectrum of DICER1‐related diseases. DICER1 appears to be the first gene implicated in the etiology of cERMS, cPNET, and PS. Young adulthood sarcomas and perhaps congenital malformations such as TGA may also be associated. 32:1381–1384, 2011. ©2011 Wiley Periodicals, Inc.

Germline DICER1 mutations and familial cystic nephroma

Background Multilocular cystic nephroma (CN) is a benign kidney tumour and is part of a family of kidney neoplasms including cystic partially differentiated nephroblastoma and Wilms tumour (WT). CN is rarely familial or bilateral, but it occurs in about 10% of families where pleuropulmonary blastoma (PPB) is present. Recently, germline mutations in DICER1 were found in familial PPB. Objective To search for DICER1 mutations in two families with familial CN; PPB was present in one family. Additionally, to test germline DNA from 50 children with sporadic WT for DICER1 mutations. Results Both families with multiple CN were found to have mutations in DICER1 leading to premature stop codons, predicted to result in loss of the ribonuclease and dsRNA binding domains. These domains are essential to the function of DICER1. No germline mutations were found in any of the 50 children who had developed WT. Conclusion It has been established that DICER1 mutations cause familial CN and may be implicated in bilateral CN. No germline mutations were found in the patients with WT, suggesting that DICER1 mutations are unlikely to have a major role in the aetiology of sporadic WT. These results provide further evidence implicating miRNA dysregulation in tumourigenesis.

Exploring the Association Between DICER1 Mutations and Differentiated Thyroid Carcinoma

CONTEXT Carriers of germline DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 syndrome. Thyroid abnormalities are a common finding in DICER1 syndrome with multinodular goiter frequently present in many families in which a germline DICER1 mutation is segregating. Differentiated thyroid carcinoma (DTC) is infrequently seen in such pedigrees. In addition to germline DICER1 mutations, specific somatic mutations have been identified in the DICER1 ribonuclease IIIb catalytic domain in several tumor types. OBJECTIVE We aimed to determine whether such characteristic somatic DICER1 mutations are present in DTCs that arise within germline DICER1 mutation carriers. DESIGN AND SETTING The study involved an opportunistic collection of 3 cases of DTC arising in individuals suspected to have DICER1 syndrome and hospital-based ascertainment and testing was implemented. RESULTS We identified somatic DICER1 mutations in 3 DTCs arising in unrelated germline DICER1 mutation carriers, all of whom had been diagnosed in infancy with pleuropulmonary blastoma (PPB), were treated with chemotherapy, exposed frequently to diagnostic radiation, and subsequently developed DTC. The somatic mutations occurred within the DICER1 ribonuclease IIIb domain, affecting highly conserved amino acid residues central to the catalytic activity of the domain. CONCLUSION This report of somatic DICER1 mutations in DTC strengthens the association between DTC and the DICER1 syndrome. The possible association between germline DICER1 mutations, PPB treatment, and the risk of subsequent DTC must be considered by clinicians when treating PPB.

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.

An immunohistochemical evaluation of C4d deposition in pediatric inflammatory liver diseases.

C4d is a marker of the activated complement cascade used to assess the humoral component of rejection, mostly in kidney allograft transplants. The role of C4d deposition has recently been addressed in hepatic allograft but has never been tested in a series of inflammatory liver diseases without previous liver transplantation. The aim of this study was to compare the immunohistochemistry profile of C4d deposition in a pediatric population, between a cohort of patients with autoimmune hepatitis (AIH) and a series of patients with chronic viral hepatitis B or C. Immunohistochemical analysis was performed on 64 liver biopsies. C4d deposition was observed in 25 (83%) of 30 AIH biopsies examined, in 6 (40%) of 15 hepatitis C biopsies, and in 17 (89%) of 19 hepatitis B biopsies. No expression of C4d was observed in 4 noninflammatory liver specimens used as negative controls. In the AIH group, a staining of the periportal sinusoids was often observed, as well as focal periductal reinforcement. Centrolobular vein staining was observed in the 3 hepatitis groups with a higher frequency in viral hepatitis B biopsies. Regardless of the etiology, lymphoid aggregates demonstrated an accentuation of the staining. These results confirm a role for a humoral immune response in pediatric autoimmune as well as in viral hepatitis. The relative ratios of positive cases imply that this immunostaining does not represent a strong diagnostic criterion in the differentiation between viral hepatitis and AIH. However, differences in the pattern of the staining were observed, depending on the etiology of the disease. The high prevalence of C4d reactivity in viral hepatitis strongly suggests that C4d does not represent a useful marker in the differentiation between acute rejection and viral hepatitis relapse in liver transplants.

SNP genotyping of a sclerosing rhabdomyosarcoma: reveals highly aneuploid profile and a specific MDM2/HMGA2 amplification.

Since the first description of sclerosing rhabdomyosarcoma in 2000, 19 pediatric cases have been reported in the literature. However, it is debated whether sclerosing rhabdomyosarcoma represents a specific rhabdomyosarcoma entity or a variant of embryonal or alveolar rhabdomyosarcoma. To date, 6 sclerosing rhabdomyosarcoma karyotypes and 1 sclerosing rhabdomyosarcoma comparative genomic hybridization profile have been reported. We present the first whole-genome tumoral genotyping of a sclerosing rhabdomyosarcoma by high-density single nucleotide polymorphism array. The single nucleotide polymorphism genotyping revealed a complex pattern including gains and losses of whole chromosomes and an amplification of the 12q13-15 region. Amplification of the 12q13-q15 region containing SAS, GLI, CDK4, and MDM2 has been observed in rhabdomyosarcoma. In the present case, the 2 amplified target genes were MDM2 and HMGA2, excluding CDK4. The identification of a specific MDM2-HGMA2 amplicon excluding CDK4 has only been described so far in well-differentiated and dedifferentiated liposarcoma. Further studies are needed to assess if this anomaly is a specific marker of sclerosing rhabdomyosarcoma.

Germ-line and somatic DICER1 mutations in a pleuropulmonary blastoma.

To the Editor: DICER1 is a non-coding small RNA processing enzyme that cleaves microRNA (miRNA) precursors into mature miRNAs, which regulate mRNA expression [1]. Germ-line DICER1 mutations are associated with the pleiotropic cancer predisposition syndrome known variously as pleuropulmonary blastoma (PPB)-familial tumor and dysplasia syndrome, and DICER1 syndrome [2–4]. This syndrome comprises several very rare entities, including PPB, cystic nephroma, Sertoli-Leydig cell tumors (SLCT), multinodular goiter, embryonal rhabdomyosarcomas (ERMS), and Wilms tumors (WT). Specific somatic mutations affecting the DICER1 RNase III domains (that affect metal ionbinding capacity) occur in trans with these germ-line mutations in persons diagnosed with SLCT, ERMS or WT [5–6]. These somatic mutations significantly reduce the production of miRNAs derived from the 5’arm (5p) of precursor miRNA, altering the function of these post-transcriptional regulators [7]. PPB is a malignant tumor of the lungs and pleura that presents in early childhood, sometimes accompanied by a family history of pediatric neoplasia that fall within the DICER1 syndrome. As stated, germ-lineDICER1mutations have been observed in patients harboring PPBs, but neither loss of heterozygosity, nor somatic mutations were reported [2,3]. The phenotypically normal presentation of obligate carriers in families in which a germ-line DICER1mutation is segregating suggests a second somatic “hit” is necessary to initiate tumor development. We now report a case of a child with a PPB that harbors two DICER1 mutations. A 3-year-old, previously healthy female of Polish descent (Fig. 1A) presented with acute dyspnea and leftsided chest pain. Chest computed tomography (CT) revealed a large heterogeneous mass occupying the majority of the left hemithorax, with mass effect shifting the cardiac and mediastinal structures. Pathology report of the initial biopsy detailed features of an embryonal rhabdomyosarcoma, but was later classified as a type II PPB, because a non-malignant cyst lined by respiratory epithelium was identified in the resection specimen. At 13 years old, the patient is now cancer-free. A deleterious germ-line mutation, c.4555delG, was observed in exon 23 of DICER1 (Ambry Genetics, Aliso Viejo, CA). This encodes a protein in which the RNase IIIa domain is preserved, but both the RNase IIIb and double-stranded RNA-binding (dsRBD) domains are lost. We subsequently identified an acquired somatic mutation, c.5438A>G, predicted to result in p.Glu1813Gly at the protein level, in the formalin-fixed paraffin-embedded (FFPE) PPB (Fig. 1B). Sequencing of cDNA synthesized from tumor RNA confirmed the expression of this mutation (Fig. 1C). It affects a highly conserved amino acid residuewithin the RNase IIIb catalytic domain, and is predicted to be damaging by both SIFT and Polyphen2 with scores of 0 and 1 respectively. The essential role of DICER1 in normal lung development is well-characterized. The acquisition of a second “hit” in the lung tissue is therefore strongly suggestive of a key mutational event capable of initiating tumorigenesis. Clinically, the potentially fatal nature of PPB argues that screening of DICER1 mutation carriers should be offered during the years of susceptibility. Identification of all at-risk children is therefore an important part of care of families of children diagnosed with PPB.

A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis

INTRODUCTION Calretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprungs disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. METHODS Records of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. RESULTS In the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. CONCLUSION The use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

High-sensitivity sequencing reveals multi-organ somatic mosaicism causing DICER1 syndrome

Background Somatic mosaicism is being increasingly recognised as an important cause of non-Mendelian presentations of hereditary syndromes. A previous whole-exome sequencing study using DNA derived from peripheral blood identified mosaic mutations in DICER1 in two children with overgrowth and developmental delay as well as more typical phenotypes of germline DICER1 mutation. However, very-low-frequency mosaicism is difficult to detect, and thus, causal mutations can go unnoticed. Highly sensitive, cost-effective approaches are needed to molecularly diagnose these persons. We studied four children with multiple primary tumours known to be associated with the DICER1 syndrome, but in whom germline DICER1 mutations were not detected by conventional mutation detection techniques. Methods and results We observed the same missense mutation within the DICER1 RNase IIIb domain in multiple tumours from different sites in each patient, raising suspicion of somatic mosaicism. We implemented three different targeted-capture technologies, including the novel HaloPlexHS (Agilent Technologies), followed by deep sequencing, and confirmed that the identified mutations are mosaic in origin in three patients, detectable in 0.24–31% of sequencing reads in constitutional DNA. The mosaic origin of patient 4s mutation remains to be unequivocally established. We also discovered likely pathogenic second somatic mutations or loss of heterozygosity (LOH) in tumours from all four patients. Conclusions Mosaic DICER1 mutations are an important cause of the DICER1 syndrome in patients with severe phenotypes and often appear to be accompanied by second somatic truncating mutations or LOH in the associated tumours. Furthermore, the molecular barcode-containing HaloPlexHS provides the sensitivity required for detection of such low-level mosaic mutations and could have general applicability.