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, Nicholas Gottardo8, Ursula R. Kees9, Surya P. Rednam10,

Liselotte P. van Hest11, Marjolijn C. Jongmans12, Shalini Jhangiani13, James R. Lupski13, 14,

Margaret Zacharin15, Dorothée Bouron-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 the Department of Medical Genetics, The Lady Davis

Institute, Segal Cancer Centre, Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal,

QC, Canada, H3T 1E2. Email: william.foulkes@mcgill.ca.

SUPPLEMENTARY FIGURES

Supplementary Figure S1:

Supplementary Figure S1 Legend:

Flow chart 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 domain occurring in PinB and

other DICER1-associated tumour types.

Supplementary Table S1: 2x2 Contingency Table - The occurrence of RNase IIIb missense mutations in PinB vs RNase IIIb missense mutations in other DICER1-associated tumours

Somatic missense mutation in DICER1 RNase IIIb domain

No somatic missense mutation in DICER1 RNase IIIb domain

PinB 0 6

Other DICER1-associated tumours 59 1

Other DICER1-associated tumours included were: Pituitary blastoma (PitB), pleuropulmonary blastoma (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 that the 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 two DICER1 “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 Doros et 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 DICER1 mutations. LOH has been identified in two cases of pituitary blastoma

[3] but only the one case which harboured both a germ-line  DICER1 mutation 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