SUPPLEMENTARY INFORMATION
Fluorescence in situ hybridization (FISH)
FISH was used to confirm the CNAs found by SNP array and detection of the ‘pre-leukaemic cell’ in the healthy child of a pair of twins discordant for Hyperdiploid ALL. In twin pair 2 Chromosome Enumeration Probes (CEP) probes (Vysis) were obtained for chromosome X and 18 and fosmid clones (BACPAC Resource Centre, Children’s Hospital, Oakland Research Institute, Oakland, CA. for CDKN2A (G248P82010F15) gene. The fosmid probe was labeled with biotin-16-dUTP, hybridised and detected with streptavidin Cy5. Fluorescent signals were viewed using a Zeiss Axioskop Fluorescent microscope equipped with epifluorescence and filters for DAPI (4’,6-diamidino-2-phenylindole) and FITC (fluorescein isothiocyanate), Spectrum Orange and Cy5. Images were captured and analyzed using a charge couple device (Photometrics) and SmartCapture Software (Digital Scientific). For the discordant twin pair 3 the same CEP probes were used for chromosome X and 18 with a BAC probe for TCF3gene (RP11-317H11). This BAC probe was labeled and detected in the same way as the fosmid probe used. The fosmid/BAC probes were tested on normal metaphase spreads to confirm the correct chromosome localization1.
Cut-off levels (%) for scoring genetically distinctive sub-clones were determined using normal blood controls as described previously.2 We scored 100 normal male cells for the presence of the signal pattern corresponding to 3 copies of chromosome 18, 2 copies of chromosome X and 2 copies of TCF3 (signal pattern 3R 2G 2P seen in healthy co-twin 3b). No cells with this signal pattern were observed. No cells with the signal pattern 3R 2G (corresponding to the hyperdiploid clone) were observed in a further 200 normal male cells. No cells with the signal pattern 3R 2G 3Aqua (control for the aberrant gain of a signal in a hyperdiploid cell), were seen in a further 800 normal male cells. In this case the Spectrum Aqua fluorochrome (Vysis) was substituted for Cy5 to aid FISH scoring.
Supplementary Tables
Supplementary Table 1: Clinical and cytogenetic details of children with Hyperdiploid ALL
Presenting WCC / Sex / Cytogenetic analysis of leukaemia at diagnosis / FISH/RT-PCRTwin 1a / 7.1 / M / 54,XY,+X,+4,+6,+8,+14,+17,+21,+21[7]/46,XY[3] / Extra AML1 and IGH
Twin 1b / 44 / M / 54,XY,+X,+4,+6,+8,+14,+17,+21,+21[8]/46,XY[2] / Extra AML1 and IGH
Twin 2a / 163 / M / 56,XY,+X,+Y,+4,+6,+10,+14,+17,+18,+21,+21[13]/ 57,idem,+21[2] / Monoallelic deletion of CDKN2A (31%). Extra signals observed for AML1 consistent with +21, +21 (73%) / idem,+21 (23%)
Twin 2b / 15.6 / M / 56~57,XY,+X,+Y,+4,+6,+10,+14,+17,add(17)(p1),+18,+21,+21,inc[cp10] / CDKN2A FISH showed a 1R2G0F signal pattern in 7% cells, Extra signals observed for AML1 consistent with +21, +21 (72%) / idem,+21 (22%). +17, +4, +6, +Y
Twin 3a / 1.2 / M / 57, XY, +X, +Y, +4, +6, +8, +10, +14, +14, +18, +21+21,add (22)(p11)[3]/58, idem, +7[1]/58,idem, +7, der(19)t(1; 19)(q21; p13)[13]/46,XY[2] / Not performed
Supplementary Table 2: Primers used for mutation analysis in Hyperdiploid ALL patients3
Analysis / Primer SequenceNRAS exon 2 (codons 12 and 13) / Forward 5’-gtactgtagatgtggctcgcca-3’
Reverse 5’-gcctcacctctatggtgggat-3’
NRAS exon 3 (codon 61) / Forward 5’-acccccaggattcttacagaa-3
Reverse 5’-gcctgtcctcatgtattggtct-3’
KRAS exon 2 (codons 12 and 13) / Forward 5’-tgtattaaccttatgtgtgacatgttc-3’
Reverse 5’–caccagtaatatgcatattaaaacaag-3’
KRAS exon 3 (codon 61) / Forward 5’ –ctgtgtttctcccttctcaggattc-3’
Reverse 5’-aagaaagccctccccagtcct-3’
PTPN11 exon 3 / Forward 5’-ccgacgtggaagatgagatctg-3’
Reverse 5’-catacacagaccgtcatgcatttc -3’
PTPN11 exon 13 / Forward 5’-ctctgagtccactaaaagttgtgcat-3’
Reverse 5’-agcaagagaatgagaatccgca-3’
Supplementary Table 3: Presence and size (in Mb) of SNP-defined CNA and associated genes in twin pairs
Twin set 1 / Twin set 2Cytoband / 1a / 1b / 2a / 2b / Gene(s) in region / Reported in Karyotype*
Whole Chr. Gains
4 / +1 / +1 / +1 / +1 / Yes
6 / +1 / +1 / +1 / +1 / Yes
7 / Yes
8 / +1 / +1 / Yes
10 / +1 / +1 / Yes
12 / Yes
14 / +1 / +1 / +1 / +1 / Yes
17 / +1 / +1 / +1 / +1 / Yes
18 / +1 / +1 / Yes
21 / +2 / +2 / +2 / +2 / Yes
22 / Yes
X / +1 / +1 / +1 / +1 / Yes
Y / +1 / +1 / Yes
Deletions
9p21.3 / 15.3 / CDKN2A+2B / No
9p13.2 / 10.1 / PAX5+ZCCH7 / No
9p24.1 / JAK2 / No
10q23.31-25.1 / 23.1 / PTEN+ADD3 +others / No
13q12.13 / 0.13 / USP12/FLT3 / No
Xq21.32 / 1.01 / NAP1L3/FAM133A / No
*Abnormality reported by conventional karyotyping and/or FISH.
Supplementary Table 4: Frequency of D835 FLT3, NRAS, KRAS and PTPN11 mutations
Mutation status*Twin set 1 / Twin set 2
Mutation tested / 1a / 1b / 2a / 2b
D835 FLT3 / WT / WT / WT / WT
KRAS / WT / WT / WT / WT
NRAS / WT / c13, GGTGAT / WT / WT
PTPN11 / WT / WT / WT / WT
* WT – Wild Type
Supplementary Table 5: Statistics of mutations found in Twin pair 2
Twin 2a / Twin 2bTotal variants / 614,307 / 595,634
On target variants / 50,588 / 50,287
Novel variants / 7,893 / 7,782
Somatic novel variants / 700 / 805
Somatic novel variants in coding region / 583 / 671
Somatic novel variants in coding region and passed GATK filter / 5 / 12
Gene with identical mutations / 1 (SLC44A2)
(19:10747230, C/T) / 1 (SLC44A2)
(19:10747230, C/T)
Supplementary Table 6: Mutations found in Twin pair 2a and b, by exome sequencing. Possible ‘Driver’ mutations in bold.
Mutation / Gene(ref) / Twin 2a / Twin 2b1_152190003_C/A / HRNR4 / Present / WT
19_10747230_C/T / SLC44A2 / Present / Present
10_26506539_C/T / GAD2 / Present / WT
2_89197301_-/TCC / IGKV5-2 / Present / WT
8_68995534_G/T / PREX25 / Present / WT
18_76754683_G/A / SALL36 / WT / Present
X_76855998_C/G / ATRX7 / WT / Present
X_70602871_C/T / TAF18 / WT / Present
6_34497338_G/A / PACSIN1 / WT / Present
17_3417225_C/A / SPATA22 / WT / Present
17_3417225_C/A / TRPV3 / WT / Present
10_129231690_C/T / DOCK19 / WT / Present
14_50655227_C/A / SOS210 / WT / Present
22_25435139_C/T / KIAA1671 / WT / Present
1_223441870_G/A / SUSD4 / WT / Present
12_75737601_G/T / GLIPR1L1 / WT / Present
WT, wild type (unmutated)
Supplementary Table 7: Specific IG/TCR gene rearrangements detected in the twins
Rearrangements / Junction*(V/N1&P/D) (D/N2&P/J) / Sequence† / Cross-test in co-twin‡
Twin 1a / IGHV1-69*02/IGHD2-2*02/IGHJ4*02 / (-7/5/-1)(-24/4/-8) / CTGTGgaaggGGATATctccGACTA
/ NA
Twin 1a / IGHV3-33*01/IGHD7-27*01/IGHJ4*02 / (0/22/-2)(0/19/-5) / AGAGAtagttcttaggact¶gtggtct¶cAACTGGGGAtctctctgggcaaagcttgTTTGA
/ NA
Twin 1b / IGHV1-69*02/IGHD2-2*02/IGHJ4*02 / (-7/5/-1)(-24/4/-8) / CTGTGgaaggGGATATctccGACTA
/ NA
Twin 1b / IGHV3-33*01/IGHD7-27*01/IGHJ4*02 / (0/22/-2)(0/19/-5) / AGAGAtagttcttaggacc¶gtggtcc¶cAACTGGGGAtctctctgggcaaagcttgTTTGA
/ NA
Twin 1b / IGLV3-12*02/IGLJ1*01 / (-2/0/-1) / ATCAT TATGT
/ Negative
Twin 2a / IGHV3-43*01/IGHD4-23*01/IGHJ3*02 / (-1/7/-10)(-4/1/-4) / AAGATccaaaaaGGTAAaGCTTT
/ NA
Twin 2a / IGKV2-30*01/Kde / (0/10/-3) / CCTCCgcaagtgaatGAGCC
/ NA
Twin 2a / IRSS/Kde / (-2/0/-5) / CCTGA GCCCT
/ NA
Twin 2a / TRBD2/TRBJ2-3 / (0/8/-21) / GAGGGaggctacgGGCCC
/ Negative
Twin 2a / TRDV2*03/TRDD3*01 / (-3/2/0) / GTGACtcACTGGGGGATACG
/ Negative
Twin 2a / TRDV2*03/TRDD3*01 / (-3/12/0) / ACTCCtctggttccACTGGGGGATACG
/ Negative
Twin 2a / TRDV2*03/TRDD3*01 / (-12/7/0) / ACTGTccgtcacACTGGGGGATACG
/ NA
Twin 2b / IGHV3-43*01/IGHD4-23*01/IGHJ3*02 / (-1/7/-10)(-4/1/-4) / AAGATccaaaaaGGTAAaGCTTT
/ NA
Twin 2b / IGKV2-30*01/Kde / (0/10/-3) / CCTCCgcaagtgaatGAGCC
/ NA
Twin 2b / TRBD2/TRBJ2-3 / (0/7/-8) / GAGGGgccggggTACGC
/ Negative
Twin 2b / TRGV2*01/TRGJP1*01 / (-3/11/-9) / GGGACcccagggggttGTTGG
/ Negative
Twin 2b / TRDV2*03/TRDD3*01 / (-12/7/0) / ACTGTccgtcacACTGGGGGATACG
/ NA
*Junction analyses were performed using the Ig BLAST ( and/or the IMGT tools ( V and J regions, D regions and N&P bases are indicated with capitals, italicized capitals and lowercase letters, respectively. ‡ Diagnostic DNA of the co-twin (sibling) was amplified using Taqman assays specific for rearrangements detected during the screening. § NA not analyzed since the rearrangement was already found to be shared during the first screening (rearrangements indicated with bold) or it was not possible to design an assay with sufficient efficiency. ¶ Single base pair differences in N1 region were found between the siblings. Rearrangements shared by the siblings are indicated with bold.
Supplementary Table 8: Copy Number abnormalities found by SNP array in Twin 3a.
Cytoband / Start base pair (Mb) / End base pair (Mb) / Size (Mb) / Candidate GenesDeletions
8q24.3 / 143.0 / 146.2 / 3.2 / Many genes
12q21.31-q21.33 / 86.9 / 89.6 / 2.7 / Many genes
19p13.3 / 0 / 5.42 / 5.42 / Many genes incl. TCF3
Gains
17q21.2-q24.3 / 39.1 / 69.8 / 30.7 / Many genes
Whole Chr. gains
+4, +6, +7. +8, +10, +14, +14 +18, +21, +21, +X and +Y
Supplementary Information References
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