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Supplementary information
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Whole exome sequencing identifies a novel somatic mutation in MMP8 associated
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with a t(1;22)-acute megakaryoblastic leukemia
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Yeunhee Kim PhD1,10, Vincent P. Schulz PhD2, Noriko Satake MD3, Tanja A.
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Gruber4,5, Alexandra Teixeira6,10, Stephanie Halene MD7, Patrick G. Gallagher MD2,8,
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and Diane S. Krause MD, PhD1,9,10*
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1Departments
of Laboratory Medicine, 2Pediatrics, 6Pathology, 7Internal Medicine
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(Division of Hematology and Yale Comprehensive Cancer Center), 8Genetics, and
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9Cell
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Haven, CT.
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3Department
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Davis, Comprehensive Cancer Center, Sacramento, CA
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4Departments
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Memphis, TN
Biology; 10Yale Stem Cell Center; Yale University School of Medicine, New
of Pediatrics (Section of Hematology/Oncology), University of California,
of Oncology and 5Pathology, St. Jude Children’s Research Hospital,
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*Correspondence to:
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Diane S. Krause
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P.O. Box 208035, 333 Cedar Street, New Haven, CT 06520-8035
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Office (203) 737-1678
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Fax (203) 785-4305
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Email: diane.krause@yale.edu
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Running title: MMP8 mutation in AMKL
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Supplementary Materials and Methods
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Sample collection
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Parental consent was obtained and documented for all procedures according to the
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University of California, Davis Internal Review Board protocol. Samples of bone
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marrow and peripheral blood were obtained from the patient at presentation with
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leukemia and on several post-induction days when the patient was in remission.
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Morphological and immunophenotypic analyses were performed at the UC Davis
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Pathology Department according to the standard protocols. FISH and cytogenetic
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analyses were performed by the Mayo Clinic Laboratory (Children’s Oncology Group
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designated laboratory). The remaining diagnostic sample was frozen. Peripheral
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blood was also collected and cryopreserved 6 and 9 months after the patient achieved
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remission.
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DNA Preparation
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The leukemic genomic DNA was extracted using the Gentra Puregene kit (Qiagen)
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according to the manufacturer’s instructions from 2.2 million acetic acid/methanol-fixed
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peripheral blood cells remaining after diagnostic FISH was performed. The Gentra
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Puregene kit provides an anionic cell lysis detergent in the presence of a DNA
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stabilizer to enhance DNA quality and quantity from archived tissue samples. The
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remission genomic DNA was extracted using the DNeasy blood and tissue kit
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(Qiagen) from thawed peripheral blood cells according to manufacturer’s instructions.
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The GenomePlex Complete whole genome amplification kit (Sigma, St. Louis, MO) (1)
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was used to generate genomic DNA. Control human genomic DNA was provided by
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the manufacturer and used as a positive control for WGA and droplet digital PCR.
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PCR analysis of t(1;22) breakpoint
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PCR amplification was performed to assess the breakpoint between chromosome 1
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and 22 with the primer sets published in Mercher, et al. (2). PCR amplification was
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performed in 20 l containing 100 ng of template DNA, 0.5 M primers, Phusion HF
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buffer (1.5mg MgCl2), 200 M dNTP, 3% DMSO and 0.02 U of Phusion DNA
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polymerase (Finnzymes, Espoo, Finland). The protocol was as follows: initial
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denaturation at 98 C for 1 min; 98 C for 10 s, 60 C for 30 s, 72 C for 2 min for 35
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cycles, and then a final extension cycle of 5 min at 72 C.
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Exome Capture and Next-Generation Sequencing
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The cancer and remission genomic DNA was captured on a Nimblegen sequence
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capture human exome 2.1 M liquid phase capture system (NimbleGen, Madison, WI)
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using the manufacturer’s protocol with modifications at the Yale Center for Genome
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Analysis at Yale University. DNA was sheared and adaptors were ligated onto the
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resulting sheared fragments. Fragments were amplified using adaptor PCR primers,
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purified and hybridized to the capture oligonucleotides. Eluted and purified fragments
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were sequenced on an Illumina GenomeAnalyzer II sequencing system (Illumina, Inc.,
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San Diego, CA) as 75bp paired end reads according to manufacturer's protocols.
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Image analysis and base calling was performed by Illumina pipeline with default
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parameters.
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Bioinformatics analyses
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Illumina sequence reads were mapped to the hg19 human genome sequence using
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the BWA version 0.5.9 alignment software. Duplicate reads arising from PCR
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amplification were flagged using the Picard software (http://picard.sourceforge.net).
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Reads near to insertion/deletions were remapped using GATK software version 1.1-37
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to reduce genotype miscalling due to alignment errors. GATK software was then used
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to recalibrate the sequence base quality scores using dbSNP version 130 SNP
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information and sequence cycle and dinucleotide covariates. Genotype call
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information was generated using the samtools mpileup command
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(http://www.ncbi.nlm.nih.gov/pubmed/19505943). Only bases with a quality of 20 or
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more and reads with a mapping quality of greater than 10 were used for genotype
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calls. The genotypes of cancer and paired remission samples were compared using
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the varscan version 2.2.7 software (http://www.ncbi.nlm.nih.gov/pubmed/22300766).
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Variants were filtered to require >2% frequency and minimum coverage of 8 reads.
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Somatic SNPs were annotated with coding effect information using the SIFT server at
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http://sift.jcvi.org/www/SIFT_chr_coords_submit.html.
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Droplet Digital PCR
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Droplet digital PCR was performed using a Bio-Rad QX100 droplet digital PCR system
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(Bio-Rad, Hercules, CA) to validate the variants identified in the diagnostic versus
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remission samples. TaqMan primers and probes (Life Technologies, Grand Island,
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NY) were custom-designed for each gene, which carried fluorescent 6-FAM and VIC
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as reporter labels at the 5’ end for mutant and normal alleles, respectively, and a
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‘minor groove binder and non-fluorescence quencher (MGB/NFQ)’ as a quencher at
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the 3’ end. The TaqMan PCR reaction mixture was assembled with a 2x ddPCR
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mastermix (Bio-Rad), 20x TaqMan primer/probes (final concentrations of 900 nM and
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250 nM, respectively) and template (10 ng genomic DNA and 5x10-7 ng mutant oligos)
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in a final volume of 20 l. The PCR mixture and 60 l of droplet generation oil were
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loaded into the sample and oil wells, respectively, for each channel of an eight-
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channel droplet generator cartridge. The droplet generator generated approximately
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15000 droplets for each PCR mixture, which were transferred to a 96-well PCR plate
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(Eppendorf, Hauppauge, NY). The plate was heat-sealed with a foil seal (Eppendorf)
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and then placed on a conventional thermal cycler. Thermal cycle conditions were; 10
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m at 95 C (1 cycle), 30 s at 95 C and 60 s at 59-63 C (40 cycles), 10 m at 98 C
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(one cycle). After PCR, the 96-well PCR plate was loaded onto the droplet reader for
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analysis. QuantSoft analysis software was used to analyze the ddPCR data. The
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primers and Taqman probes for ddPCR are listed in Supplementary Table 1.
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Cell culture and transient transfection
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HEK 293T cells were maintained in DMEM (Life Technologies, Carlsbad, CA) with
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10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), 2 mM L-
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glutamine and 1x Penicillin/Streptomycin (Life Technologies). Confluent HEK 293T
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cells were transfected using LipofectamineTM 2000 Transfection Reagent (Life
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Technologies) with 24 g of either empty vector or vector containing wild-type MMP8
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or mutant MMP8. Cells were harvested 24 hours after transfection.
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Immunoprecipitation
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Transfected cells in T75 flask were washed once with 1x Phosphate buffered saline
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and then lysed using 810 l EBC medium (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.5%
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NP, 1x phosphatase and protease inhibitor cocktails (Roche, Nutley, NJ) and 90 l of
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1,10-phenanthroline (Sigma, St. Louis, MO) in 0.1 N HCl for 20 min on ice. Cells were
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collected and centrifuged at 14,000 rpm at 4 C for 10 min. The 850 l of supernatant
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from the lysed cells were incubated with 100 l blocking EBC (EBC medium + 5%
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milk) and 30 l Anti-FLAG M2 Affinity Gel (Sigma). The mixture was incubated at 4 C
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overnight. Next day, the supernatant was removed and the immunoprecipitates were
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washed three times with EBC medium. The remaining cell lysate was used as an input
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control for western blot.
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Zymography
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Collagen zymography was performed using 0.3% rat-tail collagen type 1 (BD, Bedford,
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MA) in a 7.5% polyacrylamide gel. Fifty l of Zymogram sample buffer (BioRad) was
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added to each immunoprecipitate and the mixture was incubated for 10 minutes at
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room temperature. Fifteen l was loaded onto the collagen-polyacrylamide gel. After
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electrophoresis, the gel was incubated in 50 ml 1x Zymogram Renaturation buffer
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(BioRad) for 30 min at room temperature and changed to 50 ml 1x Zymogram
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Development buffer (BioRad), which was equilibrated for 30 min at room temperature.
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The development buffer was discarded and 50 ml 1x Zymogram Development buffer
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was added and incubated overnight at 37C. The gel was stained in 0.5% Coomassie
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Blue R-250 (BioRad) for 30 min and then destained with Methanol:Acetic Acid:dH2O
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(40:10:50). Student t-test was used for statistical analysis (p<0.05).
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Western Blot Analysis
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Beta-mercaptoethanol was added to the remaining immunoprecipitates in zymogram
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sample buffer and the whole cell lysates prior to immunoprecipitation were treated with
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denaturing Laemmli sample buffer. The samples are boiled at 98C for 3 min and 15 l
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of each sample was loaded on a 10% Mini Protean TGX gel (BioRad). After
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electrophoresis, the gel was transferred onto a 0.45 m nitrocellulose membrane. The
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blot was blocked in 5% milk/0.05% Tween in TBS for 30 min at room temperature and
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then incubated with primary mouse Anti-Flag M2 antibody in the blocking solution
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(Sigma) at 4C overnight. The blot was washed in 0.05% Tween-TBS 3 times and
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then incubated in secondary anti-mouse-horseradish peroxidase antibody for 1 hour at
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room temperature. After wash, the blot was developed in Western Lightening Plus-
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ECL solution (PerkinElmer, Waltham, MA).
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Analysis of MMP8 expression in AML patients
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RNA extraction, gene expression profiling, and transcriptome sequencing data has
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been previously published (3, 4). Sequencing data is deposited in the dbGaP
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database (http://www.ncbi.nlm.nih.gov/gap) under the accession number
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phs000413.v1.p1. Affymetrix Human Exon 1.0 ST Array data for pediatric AML
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profiling has been deposited in the NCBI gene expression omnibus
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((http://www.ncbi.nlm.nih.gov/geo/) under GSE35203. Affymetrix Human U133A Array
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data for AMKL profiling has been deposited in the NCBI gene expression omnibus
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((http://www.ncbi.nlm.nih.gov/geo/) under GSE4119.
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Figure Legends
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Supplementary Figure 1. Droplet digital PCR plots of MMP8 and CDK9. (A, C) The
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raw data of ddPCR dot plots shows the signals from normal and variant allele of
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MMP8 and CDK9, respectively. The VIC and FAM signals indicate normal (WT) and
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variant allele (MT) on the Y-axis, respectively, and the event number on the X-axis
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indicates the cumulative number of droplets generated from each sample (NTC: no
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template control, WT: normal human genomic DNA, MT: synthesized oligos carrying
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variant, Leuk: the patient’s leukemic DNA at diagnosis, and Rem: the patient’s normal
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DNA at remission), each of which is separated by yellow lines. The pink line indicates
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threshold for cut off of nonspecific signals. (B, D) The copy number of normal and
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variant alleles of MMP8 and CDK9, respectively, was calculated by Poisson
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distribution. Samples ‘WT’ and ‘Rem’ showed only VIC signal whereas ‘MT’ showed
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only FAM signals for both MMP8 and CDK9. Both VIC and FAM signals were
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observed in ‘Leuk’ for MMP8 (A, B) but not for CDK9 (C, D).
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Supplementary Figure 2. Schematic diagram of MMP8 and functional analysis of
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MMP8G189D.
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(A) Diagram of MMP8 in a 3D-ribbon representation. Green and grey dots represent
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calcium and zinc ions, respectively. (Jmol: an open-source Java viewer for chemical
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structures in 3D. http://www.jmol.org/) (B) Schematic diagram of MMP8 structural
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domains showing the variation as a red asterisk. P; leading sequences, PGBD;
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proteoglycan binding domain, H; hinge domain. (C) The conservation alignment view
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of multiple vertebrates over the variation of MMP8 (Chr11:102 592 138-102 592 237).
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Top and bottom red asterisk mark the nucleotide and amino acid variation,
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respectively.
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Supplementary Figure 3. MMP8 Expression Levels in Acute Megakaryoblastic
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Leukemia. (A) Pediatric AML cases were subjected to gene expression profiling using
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Affymetrix Human Exon 1.0 ST Arrays. Samples were clustered using average
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linkage. All samples had >80% purity, AMKL cases were all >90% pure. A detailed
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description of the samples included in this analysis can be found at NCBI gene
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expression omnibus, accession GSE35203. Briefly, AML M7 denotes AMKL
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specimens; CN, cytogenetically normal AML; M3, Acute Promyelocytic Leukemia;
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MLL, Mixed Lineage Leukemia rearranged AML; AE, AML1-ETO AML; inv16, CBFB-
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MYH11 AML. Samples with no distinguishing characteristics on cytogenetics are
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labeled as AML without modifiers. (B) A single AMKL case, SJAMLM7007
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demonstrated robust MMP8 expression by transcriptome sequencing. MMP8 exons
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are indicated, the Y-axis shows the number of supporting reads. (C) AMKL cases
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were subjected to gene expression profiling using Affymetrix Human U133A
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Arrays. Samples were clustered using average linkage. Sample purity ranged from
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12-94%. A detailed description of the samples included in this analysis can be found
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at NCBI gene expression omnibus, accession GSE4119. Briefly, M7 Ped denotes
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pediatric non-Down syndrome AMKL specimens; M7 DS, Down syndrome AMKL; M7
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Adult, adult AMKL.
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