SUPPLEMENTARY INFORMATION

Germ-line and somatic DICER1 mutations in pineoblastoma

Leanne de Kock1,2, Nelly Sabbaghian2, Harriet Druker3, Evan Weber4, Nancy Hamel4,5, Suzanne Miller6, Catherine S. Choong7, NicholasGottardo8, Ursula R. Kees9, Surya P. Rednam10, Liselotte P. van Hest11, Marjolijn C. Jongmans12, Shalini Jhangiani13, James R. Lupski13, 14, Margaret Zacharin15, DorothéeBouron-Dal Soglio16, Annie Huang17, John R. Priest18, Arie Perry19, Sabine Mueller20, Steffen Albrecht21, David Malkin22, Richard G. Grundy6 and William D. Foulkes1,2,4,5

Correspondence:Dr. William D. Foulkes at theDepartment of Medical Genetics, The Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, QC, Canada, H3T 1E2. Email:.

SUPPLEMENTARY FIGURES

Supplementary Figure S1:

Supplementary Figure S1 Legend:

Flowchart summarizing the mode of ascertainment of cases, sample acquisition, molecular analysis and the results of the study. In the section 2 (sample acquisition), boxes shaded in grey indicate samples that were sequenced by us as a part of the study. Molecular screening of samples contained in white boxes was performed at other institutions, including at the 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)

Supplementary Figure S2:

Supplementary Figure S2 Legend:Loss extends to a large region of 14q in tumours showing loss of heterozygosity.

Analysis of short tandem repeat (STR) markers for each tumour included one STR marker near DICER1 and one marker closer to the centromere of chromosome 14 q. A) Genotype analysis of STR markers visualized using gel electrophoresis of radiolabeled PCR amplicons. The two alleles are referenced for each marker using black (allele 1) and red (allele 2) bars. In tumours where there is LOH of one allele compared to the normal cells, the missing allele is always allele 2 (red). N = normal; T = tumour. B) Graphical representation of the position of the STR markers analyzed and DICER1 on chromosome 14q. Marker positions and LOH status for each patient analyzed are displayed to the right of the graphic for each marker. No = no LOH; NI = not informative (homozygous in normal tissue); LOH = loss of heterozygosity.

SUPPLEMENTARY TABLES

Supplementary Table S1: The comparison of one somatic missense mutation in DICER1 RNase IIIb domain versus no somatic missense mutation in DICER1 RNase IIIb domainoccurring in PinB and other DICER1-associated tumour types.

Supplementary Table S1: 2x2 Contingency Table - The occurrence of RNase IIIb missense mutations in PinB vs RNaseIIIb missense mutations in other DICER1-associated tumours
Somatic missense mutation in DICER1 RNase IIIb domain / No somatic missense mutation inDICER1 RNase IIIb domain
PinB / 0 / 6
Other DICER1-associated tumours / 59 / 1
Other DICER1-associated tumours included were: Pituitary blastoma (PitB), pleuropulmonaryblastoma (PPB), cystic nephroma(CN),Sertoli-Leydig cell tumours (SLCT) and Wilms tumour (WT)

Fishers Exact Test Results: The two-tailed P value is 7.7x10-8 indicating thatthe absence of missense RNase IIIb mutations in PinB is unlikely to be a chance finding.Cases included in the calculations were those found to possess twoDICER1“hits.” The six PinBs which were incorporated in the calculations included the one previously reported case [1], one case from the International PPB Registry (IPPBR) described by Doroset al. [2], case 10, case 11 and case 19 from the current study which harboured a germ-line DICER1 mutation and LOH of the DICER1 locus, and case 12 which carried two truncating DICER1mutations. LOH has been identified in two cases of pituitary blastoma [3] but only the one case which harboured both a germ-lineDICER1mutation and LOH was included in the calculation.

References:

PinB

1. Sabbaghian N, Hamel N, Srivastava A, Albrecht S, Priest JR, Foulkes WD (2012) Germline DICER1 mutation and associated loss of heterozygosity in a pineoblastoma. J Med Genet 49 (7):417-419. doi:10.1136/jmedgenet-2012-100898

2. Doros L, Schultz KA, Stewart DR, Bauer AJ, Williams G, Rossi CT, Carr A, Yang J, Dehner LP, Messinger Y, Hill DA (1993) DICER1-Related Disorders. In: Pagon RA, Adam MP, Ardinger HH et al. (eds) GeneReviews. University of Washington, Seattle, Seattle WA,

PitB:

3.de Kock L, Sabbaghian N, Plourde F, Srivastava A, Weber E, Bouron-Dal Soglio D, Hamel N, Choi JH, Park SH, Deal CL, Kelsey MM, Dishop MK, Esbenshade A, Kuttesch JF, Jacques TS, Perry A, Leichter H, Maeder P, Brundler MA, Warner J, Neal J, Zacharin M, Korbonits M, Cole T, Traunecker H, McLean TW, Rotondo F, Lepage P, Albrecht S, Horvath E, Kovacs K, Priest JR, Foulkes WD (2014) Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations. Acta Neuropathol. doi:10.1007/s00401-014-1285-z

Sahakitrungruang T, Srichomthong C, Pornkunwilai S, Amornfa J, Shuangshoti S, Kulawonganunchai S, Suphapeetiporn K, Shotelersuk V (2014) Germline and Somatic DICER1 Mutations in a Pituitary Blastoma causing Infantile Onset Cushing Disease. The Journal of Clinical Endocrinology & Metabolism 0 (0):jc.2014-1016. doi:doi:10.1210/jc.2014-1016

PPB:

Foulkes unpublished data.

Murray MJ, Bailey S, Raby KL, Saini HK, de Kock L, Burke GA, Foulkes WD, Enright AJ, Coleman N, Tischkowitz M (2014) Serum levels of mature microRNAs in DICER1-mutated pleuropulmonary blastoma. Oncogenesis 3:e87. doi:10.1038/oncsis.2014.1

Seki M, Yoshida K, Shiraishi Y, Shimamura T, Sato Y, Nishimura R, Okuno Y, Chiba K, Tanaka H, Kato K, Kato M, Hanada R, Nomura Y, Park MJ, Ishida T, Oka A, Igarashi T, Miyano S, Hayashi Y, Ogawa S, Takita J (2014) Biallelic DICER1 mutations in sporadic pleuropulmonary blastoma. Cancer Res. doi:10.1158/0008-5472.CAN-13-2470

Pugh TJ, Yu W, Yang J, Field AL, Ambrogio L, Carter SL, Cibulskis K, Giannikopoulos P, Kiezun A, Kim J, McKenna A, Nickerson E, Getz G, Hoffher S, Messinger YH, Dehner LP, Roberts CW, Rodriguez-Galindo C, Williams GM, Rossi CT, Meyerson M, Hill DA (2014) Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences. Oncogene 0. doi:10.1038/onc.2014.150

CN:

Foulkes unpublished data

Doros LA, Rossi CT, Yang J, Field A, Williams GM, Messinger Y, Cajaiba MM, Perlman EJ, K AS, Cathro HP, Legallo RD, Lafortune KA, Chikwava KR, Faria P, Geller JI, Dome JS, Mullen EA, Gratias EJ, Dehner LP, Hill DA (2014) DICER1 mutations in childhood cystic nephroma and its relationship to DICER1-renal sarcoma. Mod Pathol. doi:10.1038/modpathol.2013.242

SLCT:

Heravi-Moussavi A, Anglesio MS, Cheng SW, Senz J, Yang W, Prentice L, Fejes AP, Chow C, Tone A, Kalloger SE, Hamel N, Roth A, Ha G, Wan AN, Maines-Bandiera S, Salamanca C, Pasini B, Clarke BA, Lee AF, Lee CH, Zhao C, Young RH, Aparicio SA, Sorensen PH, Woo MM, Boyd N, Jones SJ, Hirst M, Marra MA, Gilks B, Shah SP, Foulkes WD, Morin GB, Huntsman DG (2012) Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers. N Engl J Med 366 (3):234-242. doi:10.1056/NEJMoa1102903

WT:

Wu M, Sabbaghian N, Xu B, Addidou-Kalucki S, Bernard C, Zou D, Reeve A, Eccles M, Cole C, Choong C, Charles A, Tan T, Iglesias D, Goodyer P, Foulkes W (2013) Biallelic DICER1 mutations occur in Wilms tumours. J Pathol. doi:10.1002/path.4196