© 2010 Nature America, Inc. All rights reserved. contributed contributed equally to this work. should Correspondence be addressed to J.H.J. ([email protected]). of Tumorimmunology, Radboud University Nijmegen Medical Centre and Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands. Nijmegen, The Netherlands. Nijmegen, The Netherlands. 1 (UPN) numbers patent unique indicate boxes the in Numbers indicated. are domains SANT two the and domain a C-rich domain, SET catalytic The acids. amino indicates ruler The MDS. different ten disomy. ( uniparental UPD, alterations; number contains which 7q36.1, at microdeletion a 130-kb showed 87 Subject subjects). different four represents asterisk bars, (green duplications 6 showed and bars) (red deletions carried 14 bars), (blue UPD of areas large carried Two subjects numbers. patient unique are right the on Numbers arrays. SNP high-resolution by MDS with ( 1 Figure of chromosome 5 or 5q (15%) deletions and which 7of orMDS, 7qwith (10%) individuals are of the 40–60% mostin frequent aberrations therapy. targeted of ment develop the eventually, and, of disease prognostication and fication classi proper promote may MDS in aberrations genetic underlying be cannot of the Identification disease cytopenias. the severe from die most and for achieved, cure a elderly individuals, the affected of in majority particularly malignancies, frequent most hematopoietic the of one is MDS lineages. megakaryocytic and/or by of dysplasia the characterized myeloid, disorders poietic erythroid hemato­ clonal acquired of group heterogeneous a comprise MDS modification functions strongly Analysis here The 7 In Theo de Witte Adrian van der Heijden Ruth Knops Nikoloski Gorica myelodysplastic syndromes methyltransferase gene Somatic mutations of the histone Nature Ge Nature Received 15 January; accepted 18 May; published online 4 July 2010; a Laboratory Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre and Nijmegen Centre for Molecular Life Sciences,

) Genomic aberrations of chromosome 7 detected in 102 individuals individuals 102 in 7 detected chromosome of aberrations ) Genomic or

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VOLUME 42 | NUMBER 8 | AUGUST 2010 AUGUST | 8 NUMBER | 42 VOLUME syndromes on

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Laboratory Laboratory for Genetics of Malignant Disorders, Centre for Human Genetics, University Hospital Leuven, Leuven, Belgium. Department of Human Genetics, Radboud University Nijmegen Medical Centre and Nijmegen Centre for Molecular Life Sciences, at

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3 8 / two subjects who showed UPD at 7q ( 7q at UPD showed who subjects two sequenced we mutations, homozygous acquired harbor may UPD of regions segmental large was left in the malignant bone marrow of cells of copy this individual. intact Because no that implied mutation this of presence The CUL1 as well as by the with two UPD, subjects two and genes, encompassed at 7q36.1. This region was shared by 13 out of 14 subjects with ( deletions, subjects two in (UPD) disomy uniparental to due heterozygosity of loss and subjects 102 of out 14 in deletions 7 chromosome revealed 7. on SNP genes array analysis chromosome affected to identify used MDS with individuals the of 26% in of perturbation others identified published been has lesions array genomic identify aberrations in MDS. The complete of overview SNP chrom on elusive. remained have 7 chromosome on genes implicated relevant been have genes Several codon was found in in found was codon ref. in described as nomenclature in sites of the remaining copy of sequence n 1 Fig. 1 b a g 1 . Previously, we applied SNP arrays on a cohort of 102 subjects to to subjects 102 of cohort a on arrays SNP applied we Previously, CUL1 6 2 0 and and a ). In subject 87, we found a monoallelic microdeletion of 130 kb . However, a frameshift mutation in exon 7 (703delGinsAA, 7 in exon (703delGinsAA, mutation . However, a frameshift

100 analysis of subject 87 covering the coding region and region splice the coding 87 of covering subject analysis EZH2 SANT 200 ( 87 e i r b i. 1 Fig. 1 EZH2 10 300 a ( and and EZH2 Table f CUL1 tions n o i at c i n u m m o c 81 TET2 upeetr Fg 1 Fig. Supplementary 400 3 in the bone marrow cells of the the of cells marrow bone the in . Using DNA sequencing, we and and we sequencing, DNA Using . 1 12 and 5) introducing a premature stop stop premature a introducing 5) , , 7 Supplementary Figs. 2 2 Figs. Supplementary SANT 3,4 , located , atlocated chromosome 4q24, Fig. 1 Fig. . Here, the same cohort was was cohort same the Here, . EZH2 CUL 2 148.05 Mb 50 1 73 0 a showed no aberrations C-rich ; subjects 97 and 103). 103). and 97 subjects ; 148.10 Mb UP UP UP N N N 7q36. 60 o 0 some 5, but the the but 5, some 1 148.15 Mb 96 Frameshift mutations Donor splicesitemutations Missense mutation 5 These These authors 40 SE 7q36.1 4 148.20 Mb ). Genomic Genomic ). 97 T Department Department 70 0 EZH2 96 and 10 10 s EZH2 10 97 59 53 44 76 71 58 64 28 68 26 87 75 * 3 4 3 7 1 665 UP CN 3 8 00 D A ). ).

© 2010 Nature America, Inc. All rights reserved. T cells from the same individuals. Polyclonal T Polyclonal individuals. same the from cells T DNAwe acquired, analyzed tions were of somatically non ( alleles different two two on 96, resided that found subject were in mutations point and different UPD, to was due mutation homozygous point was a that 97, found subject In other the mutation. and point deleted a was contained allele one 87, subject In biallelic. were and truncated proteins were in observed 8 out of 126 subjects (6%; EZH2 and in 24 additional individuals with MDS ( MDS, we sequenced the gene in all 102 subjects from the initial cohort ( 97 subject in present was mutation missense EZH2 a N/A, notanalyzed;chr., chromosome. T were absentinacohortof250unaffecteddonors( array analysis.To assesswhethermutationswereacquired,Tcellsfromthesameindividualsanalyzedorallelicdiscriminationassaysappliedtoshowthat deletion thatwasidentifiedontheSNParraymissedbykaryotypeanalysis.Insubject 43, the7qdeletionthatwasrevealedbykaryotypeanalysisnotdetectedaSNP compared tothereferencegenomicsequenceNT_007914.15anddeducedproteinsequenceswereNP_004447.2.Insubjects44104,7q donor andacceptorsitesrevealedmissensemutations,donor-splice-site mutations,deletionsandinsertions,predictingaminoacidsubstitutions truncations.Sequenceswere Aberrations of 127 126 121 119 114 113 112 108 106 104 103 97 96 87 81 76 73 68 64 59 58 53 44 43 40 28 26 10 7 3 UPN T B 666 73 and 40 10, subjects from cells frozen viably from generated Mutation wasabsentinacohortof250unaffecteddonors. able 3 able able 1 To assess the prevalence of of prevalence the Toassess e i r

Fig. 1 Fig. RA RA RAEB RAEB RA RAEB RARS RAEB RA RAEB-t RAEB RA RA RA RA RAEB-t RAEB RAEB-t RAEB RAEB RAEB RAEB RAEB MDS-U RAEB RAEB RA/RAEB RAEB-t RAEB-t RAEB-t FAB . UPN,uniquepatientnumber;FAB, French-American-Britishclassification;WHO, World HealthOrganizationclassification; IPSS,InternationalPrognosticScoringSystem; missense, donor mutations were not detected in subject 103, but 103, a in subject homozygous were mutations not detected

Supplementary Fig. 4 Fig. Supplementary C f haracteristics of haracteristics subjects with Classification b EZH2 tions n o i at c i n u m m o c ). In three individuals, we could show that mutations mutations that show could we individuals, three In ). RA RCMD RAEB-1 RAEB-1 RCMD RAEB-1 RCMD-RS RAEB-1 RA AML RAEB-1 RCMD RCMD RA RCMD AML RAEB-1 RAEB-2 RAEB-2 RAEB-1 RAEB-2 RAEB-1 RAEB-2 MDS-U RAEB-2 RAEB-1 RA/RAEB-1 RAEB-2 RAEB-2 RAEB-2 WHO atchromosome7q36.1weredetectedin29bonemarrowsamplesfrom126individualswithMDS.Genomicsequencingofthecodingregionandsplice - splice Low Int-2 Int-2 Int-2 Int-2 Int-2 Int-1 Int-1 Int-2 High Int-1 Int-1 Low Low Int-1 High Int-1 High High Int-2 High Int-2 Int-2 Int-2 High Int-2 Int-2 Int-2 Int-2 High IPSS - site and frameshift mutations predicting ). To investigate whether these muta these whether To ). investigate EZH2 Normal Complex withchr. 7lesions Complex withchr. 7lesions 3?3) der(7)del(7)(p11)add(7)(q del(5)(q15q33), der(7)t(1;7)(p10;q10) –7 Complex withchr. 7lesions Complex withchr. 7lesions –7 Complex Normal Normal Normal Normal Normal Complex withchr. 7lesions +8 Complex withchr. 7lesions Complex withchr. 7lesions –7 Complex withchr. 7lesions Complex withchr. 7lesions Normal del(5)(q13q33), -7 t(3;16)(q26;q2?3) +1, der(1;7)(q10;p10) del(7)(q22), del(20)(q11) Normal Complex withchr. 7lesions Complex withchr. 7lesions Karyotype mutations in individuals with with individuals in mutations S upplementary M D Supplementary TableSupplementary 1 S showing 7q36.1 aberrations or Table - cell cultures were were cultures cell M 1 ethods ). - N/A N/A N/A N/A N/A N/A N/A N/A N/A No 7q21.2–q36.3 7q11.21–q36.3 No No No No No No No No No No No No No No No No No No UPD at7q36.1 neoplastic neoplastic , S upplementary Table ). ). 1 ­

Methods ( used were primers exon 20 exon to to 11 exon 11 when or exon 13 from primers using detected were variants splice wild only 12), intron in tion splice a heterozygous harbored 73 (who In subject ( expressed was mRNA mutant the whether examined we individuals, three In ( subjects unaffected the of any 2 Table Supplementary ( (controls) individuals 250 unaffected high and allele with PCR (using assays discrimination allelic designed we 97), and 96 (subjects mutations missense carried who that ( mutations were acquired these confirming cells, T these in found were cells malignant the in ( T Supplementary Methods Supplementary No 7q36.1 No 7p21.2–q36.3 No 7q22.1 –q36.3 7q11.21–q36.3 7p22.3–q36.3 7p12.3–q36.3 7p22.3–q36.3 7p22.3 –q36.3 No No 7q11.21–q36.3 7q21.3–q36.2 No 7q31.1–q36.3 7q21.13–q36.3 7q36.1 Deletion location N/A N/A N/A N/A N/A N/A N/A N/A N/A 7q31.33–q36.3 No No able 2 SNP array and EZH2 VOLUME 42 | NUMBER 8 | AUGUST 2010 AUGUST | 8 NUMBER | 42 VOLUME - ). In subjects 10 and 127, mutant mRNA was detected, but but detected, was mRNA mutant 127, and 10 subjects In ). resolution melting analysis, HRM) and tested a cohort of of cohort a tested and HRM) analysis, melting resolution S upplementary Fig.5 Supplementary Fig. 6 Fig. Supplementary mutations

1983delA 703delGinsAA 1119-1120insC G1505+1T T2020G G2195+1A G745A DNA variation 1212-1216delGAAGA C2025A C2068T ). None of these mutations were found in in found were mutations these of None ). ). DetectedSNPsaresummarizedin ). None of the four mutations detected detected mutations four the of None ). - type mRNA was detected. No aberrant aberrant No detected. was mRNA type Table Table and and V662CfsX13 G235KfsX11 T374HfsX3 Unknown L674V Y733LfsX6 E249K variation Protein K405RfsX2 N675K R690C 1 1 Supplementary Methods Supplementary and and ). For two additional subjects subjects ). For two additional Point mutationsin Supplementary Methods Supplementary

Supplementary Fig. 5 Fig. Supplementary

Heterozygous Hemizygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Zygosity Heterozygous Homozygous Heterozygous Nature Ge Nature - donor Supplementary Supplementary - specific probes probes specific EZH2 S upplementary - site muta site N/A N/A N/A N/A N/A N/A Acquired Acquired Acquired Acquired Source n etics a a and ). ). ). ). ­ © 2010 Nature America, Inc. All rights reserved. (Tyr641; Tyr646 in NM_004456.3) leading to loss of function has has function in been of described lymphomas loss to leading NM_004456.3) in Tyr646 (Tyr641; monoallelic recurring a Recently, gene. the of function of loss to leading probably MDS, in that ( subjects affected EZH2 of in all in at one allele least or truncated altered activity was either (refs. 8–10), methyltransferase the for essential domain, SET disrupt associ here ( acids amino detected conserved modification highly mutations the epigenetic All silencing. an gene with (H3K27), ated H3 histone of lysine of 27 trimethylation and (PRC2) 2 dimethylation initiates complex complex repressive This polycomb the of unit catalytic properly. this assess to analyzed be should trial clinical prospective a from cohort larger a but sampling), marrow of bone time the from measured when 0.026 ( survival EZH2 prognosis of individuals with MDS is well known 7 Fig. any without viduals to compared indi survival worse French a showed significantly 7q deletions specific ( of MDS a classification Health WorldOrganization or System with Scoring Prognostic association International British, clear a detect transcription gene of regulation epigenetic in implicated been have proteins of family TET the and EZH2 both as notable, is This or aberrations 7q36.1 and out of eight subjects with an point mutationsand of deletions Collectively, (micro)deletions. 7q or 7 by jects the warranted. In addition to the eight subjects with are MDS with individuals from subfractions marrow bone in studies of absence or presence the of irrespective MDS with individuals of marrow bone total the in measured be could expression primary protein EZH2 in no and cells, T lines cell in protein EZH2 detect readily could we Although transcript. mutant the of stability decreased a suggesting mutant the wild than levels that lower at expressed were showed sequences analysis GeneScan and pyrosequencing Nature Ge Nature EZH2 harboring cohort The EZH2 Supplementary TableSupplementary 1 EZH2 ). The adverse effect of chromosome 7 and 7q deletions on the on the deletions 7q 7 and of chromosome effect adverse The ). point mutations result in a similar adverse tendency in overall in overall tendency adverse in a result similar mutations point encodes the histone methy histone the encodes P locus at 7q36.1 was entirely deleted at one allele in 22 sub­ 22 in at one allele deleted entirely was at 7q36.1 locus = 0.076 when measured from the date of diagnosis and is targeted by various types of deletions and mutations mutations and deletions of types various by targeted is n etics EZH2 EZH2 Fig. 1 Fig.

were present in 23% of the affected subjects. In five mutations. Therefore, more detailed expression expression detailed more Therefore, mutations. EZH2 VOLUME 42 | NUMBER 8 | AUGUST 2010 AUGUST | 8 NUMBER | 42 VOLUME EZH2 b EZH2 and and abnormalities ( abnormalities EZH2 ). Overall, ). in Overall, 40% of the subjects showing EZH2 mutations, mutations, 1 1 Supplementary Fig. 8 Fig. Supplementary point mutation in the SET domain domain SET the in mutation point . . Together with our data, this suggests Supplementary Fig. 8 Fig. Supplementary mutation, a aberrations was too small to to small too was aberrations l Table transferase that constitutes the the constitutes that transferase TET2 P 1 < 0.001; < 0.001; ). Subjects carrying 7 or carrying ). Subjects TET2 EZH2 was affected as well. well. as affected was 1 . . We show here that was present (ref. - point mutations, Supplementary Supplementary type sequences, sequences, type ). We conclude We ). conclude ). Notably, ). the - American

6–10 7–10 P = . 3 - ­ ­ .

14. 13. analysis and discrimination allelic assays. G.N. and J.H.J. wrote the paper. SNP arrays. G.N., E.R.L.T.M.T., M.M., A.v.d.H. and T.N.S. performed sequence subject material and clinical data. S.M.C.L. and R.P.K. performed and analyzed the G.N., M.M., S.M.C.L., R.K., E.R.L.T.M.T., A.v.d.H., T.N.S., P.V. and T.d.W. provided G.N., S.M.C.L., R.P.K., T.d.W., B.A.v.d.R. and J.H.J. the designed experiments. Fonds Wetenschappelijk Onderzoek Vlaanderen. 92003420) and the Stichting Vanderes (07 supported by grants from the Dutch Organization for Research Scientific (NWO, We thank R. Woestenenk and E. Kamping for technical assistance. This work was Note: Supplementary information is available on the activated. are that pathways oncogenic the and context cellular the on depending mechanisms by different tion EZH2 may by caused result defective in tumor modifications promo in epigenetic Wechanges that mechanism. postulate one into simple of that indicates deregulation reported of overexpression malignancies, various in contrast, JAK mitogenic the ing and PRC2 components may exert tumor suppressor activity by silenc that showing investigations recent with line in is This suppressor. tumor a as act may EZH2 that 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/naturegenetics/. The authors declare no competing interests.financial AUTH A C ckn O

MPETING FINANCIAL INTERESTS FINANCIAL MPETING Simon, J.A. & Lange, C.A. Lange, & J.A. Simon, A.M. Martinez, Classen, A.K., Bunker, B.D., Harvey, K.F., Vaccari, T. & Bilder, D. Bilder,K.F.,Harvey, T.& Vaccari, B.D., Bunker, A.K., Classen, R.D. Morin, J. Muller, B. Czermin, D. Reinberg, & P. Tempst, H., Erdjument-Bromage, K., Nishioka, A., Kuzmichev, R. Cao, Tahiliani,M. S.E. Antonarakis, J.T.& Dunnen, Den F.Delhommeau, S.M.C. Langemeijer, D. Haase, R.D. Brunning, 1150–1155 (2009). 1150–1155 Dev. Genes 2008). Geneva, LymphoidTissues o O wle R R C 1 4 et al. et , suggesting , that suggesting EZH2 might also act as an oncogene. This O et al. et et al. et d

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