Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree

Fernández-Martínez et al. BMC Cancer (2017) 17:146 DOI 10.1186/s12885-017-3136-5 RESEARCH ARTICLE Open Access Identification of somatic and germ-line DICER1 mutations in pleuropulmonary blastoma, cystic nephroma and rhabdomyosarcoma tumors within a DICER1 syndrome pedigree Lorena Fernández-Martínez1, José Antonio Villegas2, Íñigo Santamaría1, Ana S. Pitiot1, Marta G. Alvarado1, Soledad Fernández3, Héctor Torres3, Ángeles Paredes4, Pilar Blay4 and Milagros Balbín1* Abstract Background: DICER1 syndrome is a pediatric cancer predisposition condition causing a variety of tumor types in children and young adults. In this report we studied a family with two relatives presenting a variety of neoplastic conditions at childhood. Methods: Germ-line mutation screening of the complete coding region of the DICER1 gene in genomic DNA from the proband was performed. The presence of somatic DICER1 mutation and further alterations in driver genes was investigated in genomic DNA obtained from available tumor samples. Results: A nonsense germ-line mutation in DICER1 causing a truncated protein at the IIIb domain level was identified segregating within a family including two affected relatives who developed in one case cystic nephroma and pleuropulmonary blastoma, and rhabdomyosarcoma and multinodular goiter in the other. Additional in trans DICER1 missense somatic mutations in the IIIb DICER1 domain were found both in the cystic nephroma and in the rhabdomyosarcoma, suggesting that neoplasms in this family might arise from the unusual two-hit mechanism for DICER-derived tumorigenesis in which after the presence of a truncated constitutive protein, a neomorphic DICER1 activity is somatically adquired. Additional genetic alterations, such as TP53 mutations, were identified in the rhabdomyosarcoma. Conclusions: Besides DICER1 loss of standard activity, oncogenic cooperation of other genes, as mutated TP53, may involve developing higher grade tumors within this syndrome. Given the broad clinical spectrum that may arise, genetic counseling and close surveillance must be offered to all family members at risk of DICER1 syndrome. Keywords: DICER1 mutations, DICER1 syndrome * Correspondence: 1 Laboratorio de Oncología Molecular, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), AGC Laboratorio de Medicina, Hospital Universitario Central de Asturias (HUCA), Oviedo 33011, Spain Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fernández-Martínez et al. BMC Cancer (2017) 17:146 Page 2 of 6 Background Germ-line mutations in DICER1 have been described in the so-called DICER1 syndrome, a pleiotropic pediatric cancer predisposition condition causing a variety of tumor types in children and young adults, including pleuropulmonary blastoma (PPB), cystic nephroma (CN), rhabdomyosarcoma (RMS), multinodular goiter, ovarian Sertoli-Leydig cell tumor and other neoplastic conditions. DICER1 is a multidomain protein, containing two endoribonuclease III domains. In the majority of cases, germ-line mutations are nonsense, frameshift or splice-site mutations leading to premature truncation of the protein, resulting in loss of RNAseIII function [1–3]. RNA processing endoribonucleases are required for the biogenesis of microRNAs (miRNAs), cleaving precursor miRNAs into mature miRNAs which, in turn, post-transcriptionally regulate messenger RNA expression [2]. Disregulation of miRNAs is implicated in several human diseases, as they participate in many different biological processes. Thus, mutations in DICER1 have the potential to affect many biological functions and originate different phenotypes. In this communication, germ-line and somatic mutations in DICER1 are reported within a family with two relatives presenting a variety of neoplastic conditions at childhood. Hot spot mutations in KRAS, NRAS, EGFR, PIK3CA and BRAF were sought by real-time quantitative allelespecific PCR amplification using commercial kits (RAS Mutation Screening Panel, Entrogen, USA; therascreen EGFR RGQ PCR Kit V2, Qiagen, UK; cobas PIK3CA Mutation Test, Roche, USA; cobas 4800 BRAF V600 Mutation Test, Roche, USA), following manufacturer’s instructions. Deletions or amplifications in PDGRFA, TP53, CDKN2A, CDK4, RB1, EGFR, PTEN, and MMDM2 genes and in the chromosomal regions 1p and 19q were studied by MLPA (Multiplex Ligation-dependent Probe Amplification) using commercial kits (P0471, P088, and P105 probemixes, MRC-Holland, The Netherlands), following manufacturer’s instructions. Methods Germ-line mutational analysis Subjects Considering patient’s tumors nature, genetic mutation screening of the complete coding region of the DICER1 gene in genomic DNA from proband’s blood was performed. It led to identify a nonsense truncating mutation affecting Q1783 residue, codified in exon 24 (c.5387C > T; p.Q1783*). This mutation was found in heterozygosity and predicted to truncate the protein by the RNaseIIIb domain of the enzyme. We studied segregation of this germ-line mutation with diverse pathologies in 7 available relatives and identified the proband’s mother and grandmother as carriers of the mutation (Fig. 2). In addition, a 21-year-old female cousin of the proband who was diagnosed of an embryonal RMS (Fig. 1c) at age 14 and multinodular goiter at age 20, was also germ-line carrier of the DICER1 mutation. Interestingly, thyroid affection was also reported in most of the family members, being multinodular goiter with calcifications the only remarkable pathological phenotype present in the proband’s mother and grandmother. Of those participating in the study, 4 out of 5 affected of thyroid alterations carried the p.Q1783* mutation. Our studied pedigree comprised 8 individuals. All subjects or their parents/legal guardians gave written informed consent for genetic research studies and peripheral blood samples were taken. Available frozen tumor tissue samples were obtained from HUCA Tumor Bank. Written informed consent for sample banking and research use was obtained at the time of the surgery. DNA, RNA and cDNA samples DNA from peripheral blood and tumor tissues was extracted using DNAzol (Molecular Research Center, USA), following manufacturer’s instructions. RNA was obtained from frozen CN and ERMS frozen tissue samples with Trireagent® (Ambion). cDNA was synthesized with RNA ImProm-II Re (...truncated)