Identification of a Novel TP53 Cancer Susceptibility Mutation Through Whole-Genome Sequencing of a Patient with Therapy-Related AML

Supplementary Online Content

Link DC, Schuettpelz LG, Shen D, et al. Identification of a novel TP53 cancer susceptibility mutation through whole-genome sequencing of a patient with therapy-related AML. JAMA. 2011;305(15):1568-1576.

eFigure 1. t-AML patient’s karyotype

eFigure 2. Somatic copy number alterations and region of chromosome 17 UPD

eFigure 3. Interphase fluorescence in situ hybridization (FISH) analyses of the t(3;4) translocation

eTable 1. Somatic genic SNVs and indels in t-AML patient

eTable 2. Somatic CNAs

eTable 3. Structural variants

eMethods

This supplementary material has been provided by the authors to give readers additional information about their work.

Downloaded From: https://jamanetwork.com/ on 10/02/2021 45, XX, add(3)(q27), del(3)(q12), -4, del(5)(q12q33), -7, +add(18)(p11.1), +mar, +mar1[cp19]/46,XX[1]

Supplemental Figure 1. t-AML patient’s karyotype. Standard karyotype showing complex changes including abnormalities of chromosomes 3 and 4, monosomy 7, del(5q), and two marker chromosomes that could not be resolved by standard cytogenetic analysis.

eFigure 1. t-AML patient’s karyotype. Standard karyotype showing complex changes including abnormalities of chromosomes 3 and 4, monosomy 7, del(5q), and two marker chromosomes that could not be resolved by standard cytogenetic analysis.

Downloaded From: https://jamanetwork.com/ on 10/02/2021 Supplemental Figure 3. Somatic copy number alterations and region of chromosome 17 uniparentaldisomy (UPD). Shown are log2 plots of the ratiosequence of reads in the leukemic vs. skin DNA. Deflections below or above zero represent somatic deletions and amplifications, respectively. The region of UPD on chromosome 17 is shown in the lower right plot. The number of heterozygous SNPs that were present in the skin DNA, but absent in the leukemic DNA, perbp of10,000 sequence (loss of heterozygous SNPs, LOH) is shown.

eFigure 2. Somatic copy number alterations and region of chromosome 17 uniparental disomy (UPD) Shown are log2 plots of the ratio of sequence reads in the leukemic vs skin DNA. Deflections below or above zero represent somatic deletions and amplifications, respectively. The region of UPD on chromosome 17 is shown in the lower right plot. The number of heterozygous SNPs that were present in the skin DNA, but absent in the leukemic DNA, per 10,000 bp of sequence (loss of heterozygous SNPs, LOH) is shown.

Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 1. Somatic genic SNVs and indels in t-AML patient Cosmic Database CDS Mutation AA *mRNA Gene Chr Position Ref Var Mutation Type Change Expression Hits # Screen Cancer Types TRAPPC3 1 36376168 T C c.241-2 Splice e4-2 Present 0 22 FAAH 1 46643650 G T c.464 Missense p.G155V Present 1 85 Breast REP65 1 68677480 C A c.840 Missense p.E280D Present 0 22 GPR156 3 121369031 C T c.1983 Silent p.K661 Present 0 44 CLSTN2 3 141661120 G A c.1041 Silent p.L347 Present 3 86 Colon, skin ENSG00000 3 181362413 C T NULL NULL Present 0 0 201822 ABCG2 4 89232527 C A c.1851 Missense p.Q617H Present 0 22 SMARCA5 4 144677089 C A c.1303 Missense p.L435I Present 1 159 Skin SERINC5 5 79477754 C T c.1153 Missense p.G385S Present 0 0 SFRS18 6 99955549 C A c.2006 Missense p.R669M Present 0 0 MIOS 7 7579832 A G c.1201 Missense p.R401G Present 0 0 C9orf79 9 89690809 C T c.1587 Silent p.P529 Absent 0 22 LOC727878 10 2530907 C A c.219 Missense p.W73C NA 0 0 NUP98 11 3702986 A T c.1770 Missense p.N590K Present 8 244 Breast, kidney, skin AGBL2 11 47668815 C T c.1020 Silent p.L340 Present 0 22 ME3 11 85948435 C del c.261 Frameshift p.D87fs Present 0 44 RDH16 12 55632933 C T c.681 Nonsense p.W227* Present 0 22 TMEM132D 12 128750858 G A c.418 Silent p.L140 Present 0 22 TRPC4 13 37135841 G A c.1400 Missense p.S467L Present 1 85 Breast GPR176 15 37886664 C T c.260 Missense p.G87E Present 0 44 EEF2K 16 22144604 C G c.53 Missense p.S18C Present 1 342 Colon PKD1L3 16 70559305 C A c.2286+1 Splice e14+1 Present 0 22 ZNF624 17 16493717 CC c.66 Frameshift p.F23fs Present 1 207 Colon GRB7 17 35152745 C T c.375 Silent p.R125 Present 0 158 LRRC37A3 17 60287290 T C c.3436 Missense p.T1146A Present 0 22 MUC16 19 8933784 C T c.14662 Missense p.E4888K Present 1 23 Skin WNK3 X 54336482 T C c.1697 Missense p.D566G Present 5 380 CNS, lung, kidney ARMCX3 X 100766745 G A c.120 Silent p.E40 Present 1 62 Lung COSMIC v45 database was searched for mutations of the indicated genes in various human cancers (http://www.sanger.ac.uk/genetics/CGP/cosmic/). *t-AML myeloblasts Affymetrix Exon 1.0 array mRNA expression data; absent and present calls are based on a detection p value of <0.05 using Affymetrix Expression Console software. All SNVs and indels were validated on the 454 (Roche) sequencing platform.

Downloaded From: https://jamanetwork.com/ on 10/02/2021

eTable 2. Somatic CNAs

Chr Type Start End Length (bp) 3 Deletion 11,100,000 12,580,000 1,480,000 3 Amplification 75,780,000 75,890,000 110,000 3 Deletion 103,820,000 106,710,000 2,890,000 3 Deletion 113,850,000 114,500,000 650,000 3 Deletion 169,450,000 170,290,000 840,000 3 Deletion 170,380,000 171,620,000 1,240,000 3 Deletion 171,710,000 173,820,000 2,110,000 4 Deletion 12,040,000 13,160,000 1,120,000 5 Deletion 94,250,000 175,300,000 81,050,000 7 Deletion 18,440,000 56,470,000 38,030,000 7 Deletion 70,010,000 101,080,000 31,070,000 12 Deletion 11,700,000 22,800,000 11,100,000

Coordinates are based on the NCBI36/hg18 assembly. CNAs were validated by PCR as described previously1 or by SNP array data.

Downloaded From: https://jamanetwork.com/ on 10/02/2021

eTable 3. Structural Variants

Chr A Breakpoint A Gene A Chr B Breakpoint B Gene B Orientation A/B 3 187463047 DGKG 4 15322981 BST1 -- 3 187478715 DGKG 4 15322889 BST1 ++ 5 118435889 DMXL1 12 11690424 ETV6 ++ 3 107261142 4 16281032 LDB2 +- 3 162745162 X 145963838 -+ 11 109323397 17 25089370 SSH2 +- 11 109323397 17 25089370 SSH2 -+ 7 11995554 12 38861200 ++

Eight novel translocations were identified by whole genome sequencing, and validated by PCR and capillary sequencing around the breakpoint. Orientation is as follows: “+-“: The plus strand sequences 5’ to Chromosome A breakpoint join the plus strand sequences 3’ to Chromosome B breakpoint; “++”: The plus strand sequences 5’ to Chromosome A breakpoint join the negative strand sequences 5’ to Chromosome B breakpoint; “-+”: The plus strand sequences 5’ to Chromosome B breakpoint join the plus strand sequences 3’ to Chromosome A breakpoint; “- -“: The negative strand sequences 3’ to Chromosome B breakpoint join the plus strand sequences 3’ to Chromosome A breakpoint.

Downloaded From: https://jamanetwork.com/ on 10/02/2021

eFigure 3. Interphase fluorescence in situ hybridization (FISH) analyses of the t(3;4) translocation Interphase FISH of leukemic blasts and normal marrow cells. Dual-color break apart probes were designed for the DGKG gene. The breakpoint is indicated by the yellow line. The intact locus is represented by a single yellow dot, while rearrangements show separate green and red signals. In total, 20 patients with t-AML were analyzed using this FISH scheme, with 100 interphase cells analyzed per sample.

Downloaded From: https://jamanetwork.com/ on 10/02/2021

eMethods

Primer list: Primer sequences for TP53 genomic PCR: Primer set 1: wt=2924 bp product; deletion mutation= no product Forward: (1132-1151; exon 9) 5’-CCAGCTCCTCTCCCCAGCCA-3’ Reverse: 5’-CCTCATTCAGCTCTCGGACA-3’

Primer set 2: wt= 4169 bp product; deletion mutation= 1179 bp Forward: 5’-CTCTGGGAGGAGGGGTTAAG-3’ Reverse: 5’-CCTCATTCAGCTCTCGGACA-3’

Primer sequences for TP53 cDNA: Forward: (848-867; exon 6) 5’-GGTGCCCTATGAGCCGCCTG-3’ Reverse: (1462-1481; exon 11) 5’-GGGGGTGGGAGGCTGTCAGT-3’

Primer sequences for BST1-DGKG cDNA: Forward: (359-378; exon 1) 5’-GCGCACTGCTGAGTCCCGAG-3’ Reverse: (1433-1414, exon 10) 5’-CCTTGCTTGCGGACGCCCAT-3’

Primer sequences for DGKG-BST1 cDNA: Forward: (1409-1428; exon 10) 5’-TGCTCATGGGCGTCCGCAAG-3’ Reverse: (903-922; exon 7) 5’-TGCTGCCTTCCCCGCAGGAT-3’

Fluorescence in situ hybridization analyses Bacterial artificial chromosome (BAC) clones overlapping the DGKG gene [RP11-1118G15 (chr3: 187280861-187431522) and RP11-259B9 (chr3: 187529031-187703479)] was obtained from the BAC/PAC Resource (Oakland, California, USA). BAC DNA was isolated using the Qiagen large construct DNA purification kit (Qiagen, Valencia, CA) and probes were labeled with Spectrum Green and Spectrum Orange by nick translation (Vysis Inc, Downers Grove, Illinois, USA), following the manufacturer’s instructions. The two probes were then mixed together in equal proportions resulting in a break-apart probe mixture. A normal signal pattern would show a yellow signal where a rearrangement would display two separate signals, one green, one orange. Slides were analyzed using a fluorescence microscope, and images recorded using Cytovision software. Quantitative FISH analysis was used to confirm a normal signal pattern (2 yellow signals) versus a DGKG gene rearrangement (1 yellow/1 green/1 red signal). This procedure was carried out for 100 interphase cells in each probe experiment. A cohort of 20 patients with therapy related AML was analyzed using the above FISH scheme.

Spectral Karyotyping (SKY) Analysis Spectral karyotyping was performed using the Applied Spectral Imaging (ASI) SkyPaint™ kit for human chromosomes using the procedure recommended by the manufacturer. Probes were detected using the SkyPaint™ detection reagents as described by the manufacturer. Analysis was performed using ASI image capturing (SI 2.2) and analysis software (SkyView 1.6.1). Chromosomal abnormalities are described according to the International System for Human Cytogenetic Nomenclature2.

Glossary of terms: Paired end reads: a technique used in massively parallel sequencing where 50-100 base pairs of sequence are obtained from each end of 250-500 base pair DNA fragments. This increases both the amount of sequence and the ability to map the sequences to the human genome.

Downloaded From: https://jamanetwork.com/ on 10/02/2021

Uniparental disomy: A situation in which both copies of a chromosome, or part of a chromosome, are derived from the same parent.

Spectral karyotyping: A cytogenetic technique whereby fluorescently-labeled probes specific for each chromosome are used to simultaneously visualize each chromosome with a different color.

Fluorescence in situ hybridization (FISH): A cytogenetic technique for the detection, using a fluorescently-labeled probe, of a specific region of a chromosome.

Entrez gene identification numbers of genes described in text TP53: 7157 (http://www.ncbi.nlm.nih.gov/gene?term=7157) BRCA1: 672 (http://www.ncbi.nlm.nih.gov/gene?term=672) BRCA2: 675 (http://www.ncbi.nlm.nih.gov/gene?term=675) CHEK2: 11200 (http://www.ncbi.nlm.nih.gov/gene?term=11200) CDH1: 999 (http://www.ncbi.nlm.nih.gov/gene?term=999) BRIP1 (FANCJ): 83990 (http://www.ncbi.nlm.nih.gov/gene?term=83990) PALB2 (FANCN): 79728 (http://www.ncbi.nlm.nih.gov/gene?term=79728) STK11: 6794 (http://www.ncbi.nlm.nih.gov/gene?term=6794) ATM: 472 (http://www.ncbi.nlm.nih.gov/gene?term=472) DGKG: 1608 (http://www.ncbi.nlm.nih.gov/gene?term=1608) BST1: 683 (http://www.ncbi.nlm.nih.gov/gene?term=683) CDKN1A: 1026 (http://www.ncbi.nlm.nih.gov/gene?term=1026) MDM2: 4193 (http://www.ncbi.nlm.nih.gov/gene?term=4193) GADD45A: 1647 (http://www.ncbi.nlm.nih.gov/gene?term=1647) NOXA: 5366 (http://www.ncbi.nlm.nih.gov/gene?term=5366) BCL2: 596 (http://www.ncbi.nlm.nih.gov/gene?term=596) BAX: 581 (http://www.ncbi.nlm.nih.gov/gene?term=581) PIG3: 9540 (http://www.ncbi.nlm.nih.gov/gene?term=9540) PTEN: 5728 (http://www.ncbi.nlm.nih.gov/gene?term=5728) MDM4: 4194 (http://www.ncbi.nlm.nih.gov/gene?term=4194) CCNG1: 900 (http://www.ncbi.nlm.nih.gov/gene?term=900)

References 1. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med. Sep 10 2009;361(11):1058-1066. 2. Shaffer LG, Campbell LJ, Slovak ML, et al. ISCN (2009): Important revisions and new additions to molecular methods for copy number detection for cytogenetic analysis. Chromosome Res. Jun 2009;17:10-11.

Downloaded From: https://jamanetwork.com/ on 10/02/2021