Germ-line and somatic DICER1 mutations in pineoblastoma

Abstract

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.

Fig. 1

Flow chart summarizing the mode of ascertainment of cases, sample acquisition, molecular analysis and the results of the study. Asterisk indicates samples sequenced by us. Sequencing of gDNA not performed by us was conducted at: referring institution (n = 2), Ambry genetics (Aliso Viejo, CA, USA) (n = 1), Prevention genetics (Marshfield, WI, USA) (n = 2), or at Baylor-Hopkins Center for Mendelian genomics (Houston, TX, USA) (n = 1)

Fig. 2

Anti-DICER1 immunostaining (magnification = 20×). Immuno-reactivity for DICER1 retained: panelsa, b and c (cases 9, 14, 13, respectively); and DICER1 immuno-reactivity lost: panels d, e, f, g and h (cases 12, 8, 10, 11, 19, respectively)

Fig. 3

Graphic representation of the unfolded DICER1 protein structure (NP_001258211.1) indicating the approximate positions of the germ-line DICER1 mutations observed in the 21 PinB cases being reported. Mutations shaded in blue represent mutations that were identified within tumour gDNA, but are not confirmed to be somatic in origin. Case number indicated at the position of each mutation. DICER1 domains, defined as follows: DExD/H DexD/H box helicase domain, TRBP-BD trans-activating response RNA-binding protein binding domain, HELICc helicase conserved C-terminal domain, DUF283 domain of unknown function, Platform platform domain, PAZ polyubiquitin-associated zinc-finger domain, c.h. connector helix, RNase IIIa Ribonuclease IIIa domain, RNase IIIb ribonuclease IIIb domain, dsRBD double-stranded RNA-binding domain. Mutations: Case 8: germ-line DICER1 amino acid change, p.(Ser1585*); Case 10: germ-line DICER1 amino acid change, p.(Tyr1701*); somatic DICER1 change, loss of heterozygosity (LOH); Case 11: germ-line DICER1 amino acid change, p.(Ser1545Phefs*7); somatic DICER1 change, LOH; Case 12: DICER1 amino acid changes, p.(L1094rfs*9) and p.(Y1225X)—not confirmed to be germ-line or somatic in origin; Case 19: germ-line DICER1 amino acid change, p.(Lys500*); somatic DICER1 change, LOH; Case 20: germ-line DICER1 amino acid change, c.4050+1G>A; Case 21: germ-line DICER1 amino acid change, p.(Ser1470Leufs*19)

Fig. 4

Case 19: the proband, individual IV-4, was diagnosed with a PinB at the age of 24 years and was found to carry the germ-line mutation, c.1498A>T, in DICER1. Of the five family members tested, three were found to carry the same germ-line DICER1 mutation and all three individuals had hyperthyroidism (individuals II-2, III-2 and III-5). b Case 20: the proband, individual V-6, was diagnosed at 10 years of age with a pineoblastoma, at 15 years of age with cervical and vaginal fibroepithelial polyps, at 16 years of age with a Sertoli–Leydig cell tumour (SLCT) of the left ovary, at 17 years of age with a cervical embryonal rhabdomyosarcoma (cERMS) and a brain-stem ERMS at 21 years of age. She was found to carry the germ-line DICER1 mutation, c.4050+1G>A. Several family members had pulmonary and thyroid abnormalities. c Case 21: the proband (individual IV-3, deceased) was diagnosed at the age of 2 years with a pineoblastoma. At 6 months of age, multiple pulmonary bullae were detected and congenital bullous emphysema was diagnosed. The lung pathology was later reviewed in the light of the whole exome sequencing results and a revision of the diagnosis to pleuropulmonary blastoma (PPB) was made. Both the proband, his mother (individual III-3) and his two brothers (individuals IV-2 and IV-4) were found to carry the c.4407_4410delTTCT germ-line DICER1 mutation. The mother is affected by a multinodular goitre (MNG). Individual IV-5 was diagnosed with a meningeal sarcoma at 3 years of age. Meningeal sarcoma is not definitively associated with the DICER1 syndrome and the patient is untested

Fig. 5

Case 19 somatic analysis: aPanel I the germ-line DICER1 mutation, c.1498A>T, indicated by an asteriskPanel II loss of heterozygosity (LOH) of the wild-type allele evident at the position of the germ-line mutation (asterisk) in cDNA synthesized from tumour RNA. b The position of the germ-line DICER1 mutation predicted to truncate the protein (indicated by red arrow), relative to the anti-DICER1 antibody binding site (indicated by a yellow star)