Supplemental Figure S1. Core Biopsy Dissection Procedure for Methylight Analysis
Supplemental Figure S1. Core biopsy dissection procedure for MethyLight analysis.
Punches from paraffin-embedded tissue sample containing solely breast tumour material were taken followed by DNA isolation, bisulphite modification and MethyLight analysis.
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Nomenclature / Forward Primer Sequence / Reverse Primer Sequence / Probe Oligo SequenceBCL2 / TCGTATTTCGGGATTCGGTC / AACTAAACGCAAACCCCGC / 6FAM-ACGACGCCGAAAACAACCGAAATCTACA-BHQ-1
CACNA1G / TTTTTTCGTTTCGCGTTTAGGT / CTCGAAACGACTTCGCCG / 6FAM-AAATAACGCCGAATCCGACAACCGA-BHQ
CALCA / GTTTTGGAAGTATGAGGGTGACG / TTCCCGCCGCTATAAATCG / 6FAM-ATTCCGCCAATACACAACAACCAATAAACG-BHQ-1
CDH13 / AATTTCGTTCGTTTTGTGCGT / CTACCCGTACCGAACGATCC / 6FAM-AACGCAAAACGCGCCCGACA-BHQ-1
CRABP1 / TCGAAATTTTCGTTGTTGCGT / TATCCGTACCTACCGCCGC / 6FAM-ACCATACCCAACTTCGCCGACACCTAA-BHQ
CYP1B1 / GTGCGTTTGGACGGGAGTT / AACGCGACCTAACAAAACGAA / 6FAM-CGCCGCACACCAAACCGCTT-BHQ-1
CYP27B1 / GGGATAGTTAGAGAGAACGGATGTTT / CCGAATATAACCACACCGCC / 6FAM-CCAACCTCAACTCGCCTTTTCCTTATTTCA-BHQ-1
DCC / GGGTTCGGCGCGTGT / CGAAAAATACAAAAACCAACTTAAATACC / 6FAM-ACCAAAAATCGCGAACAACGACAACACT-BHQ-1
DLC1 / AGTAAGGATGCGTTGAGGATCG / ACGACTCGACTTCCGCGTC / 6FAM-AACCCACGACGACACCCGAAACG-BHQ-1
EBF3 (DKFZ) / GTAGGATATTGCGGGATCGTTC / GCAACACTCACTACCCCGTTTAT / 6FAM-TCTTTAAAACAAACGAACCGCGCCAA-BHQ-1
ESR1 / GGCGTTCGTTTTGGGATTG / GCCGACACGCGAACTCTAA / 6FAM-CGATAAAACCGAACGACCCGACGA-BHQ-1
FLJ39739 / GAACCCTCCGCGCCC / TGTTCGGGATGGTTGATATTTTG / 6FAM-CCGCCTAATCACCCGACCCGA-BHQ-1
GABRA2 / TCGTCGGAGGAGCGGA / AACCTCTCGAAAACCCCAACA / 6FAM-ACGACCTCGAAAAACAACCCGAAACTACG-BHQ-1
GAD1 / CGATTGGTTCGGCGTAGAAA / CCCTCCGATATACAAAACCCC / 6FAM-CCCGCACAACTCTCGCTTCTCTTTACAA-BHQ-1
GATA3 / TGTATCGGGACGGAATCGTT / ACGCGCGCTCTAACCCTT / 6FAM-AAATATAACCGCGACTCCTACCAATTCATTCG-BHQ
GATA4 / GATGGTGGTCGCGTGAAGTTA / TTCCCTCCATATACGAACTACCG / 6FAM-CCTATCCCGAATCCGTCAATCCCG-BHQ-1
GATA5 / AGTTACGTGATTTTGGTAGGTTTTGTT / TAATCCGAACTCCGCGCTA / 6FAM-CCCGTATCGTACGTCCTTATCGCCAAA-BHQ-1
GDNF / CGGTAGTTGTCGTTGAGTCGTTC / AACAACCGCCGCTACTTTAAATA / 6FAM-CGCGCGTCGCGCTCTTAACTAAAA-BHQ-1
HIC1 / GTTAGGCGGTTAGGGCGTC / CCGAACGCCTCCATCGTAT / 6FAM-CAACATCGTCTACCCAACACACTCTCCTACG-BHQ-1
HOXA1 / TTGTTTATTAGGAAGCGGTCGTC / TCGAACCATAAAATTACAACTTTCCA / 6FAM-TCGTACGCGATCAACGCCAACAATTA-BHQ-1
HOXA10 / TGTATTGATGGGTTAGGAGACGTATT / CCCACCAACCACGTTAAAACA / 6FAM-CAACTCCCGACCTTCGAACCAAAATATCG-BHQ-1
HOXA11 / TTTTGTTTTCGATTTTAGTCGGAAT / TAATCAAATCACCGTACAAATCGAAC / 6FAM-ACCACCAAACAAACACATCCACGACTTCA-BHQ-1
HOXA13 / GGTGGTAGAAGGCGAATTTTTTAG / TCGCGAACAAATACATAAATACCG / 6FAM-GACCCAACTACCGAAAAATTCAACTCCCG-BHQ-1
HOXA6 / CGAACGCCAACGACGAA / GGTTTTTGAGGCGGTTTAAGTATTAG / 6FAM-CGACCCTTTCAATAACGAACAAAT-BHQ-1
HOXA7 / CAAAACTCACTAACAAAAATCGACG / GGAGCGTTTAGTTGGCGGT / 6FAM-ACTACGTAAAACCGAACCGCCTA-BHQ-1
HOXA9 / AAAATCCGTCCCAAACGAAA / CGTTTTAGGCGGGTAGTTGTG / 6FAM-CGCCGACCCTACCAAAACACTCCAAA-BHQ-1
HOXB7 / CCTCGAAACGATCCGTATCAC / CGTGTAGAATTGGTTTGGTTTAATAAAG / 6FAM-CCCATCCTCTTCAACCCGTTACCGAA-BHQ-1
HOXC9 / TTTCTTAACGTAATCCGTCTCTATCG / ACGTTTCGAATTGATAATCGTTGAT / 6FAM-ACGCTCCCGCAATCTCTACGCGA-BHQ-1
HOXD11 / CCGCACTCGTCAAAATCGTT / TGTAATCGATGGTTTAGGTTGGTTG / 6FAM-ACTCCGCGACGTACGCGCCA-BHQ-1
HOXD12 / ATTCGTTTTCCGCAAACTTTCTAA / TGGTGTCGTCGGATCGC / 6FAM-ACATTTTCCATTAACCGACGCCTCTCGACT-BHQ-1
HOXD8 / ACCTCCGCCGACCTTAAAA / TGTCGGTTGCGATTCGTATTT / 6FAM-ACCCATAAAAACGCACTACCAAAAACTCCGT-BHQ-1
HOXD9 / ATCGAACCCCGTCGCTAACT / GGTTTGGCGTTTTCGCG / 6FAM-ACTTCCGAACCGCGAACGACGA-BHQ-1
IGF2 / GGTTGGCGCGATTATAAGAGTC / AACCGACCGAACCTCACG / 6FAM-AACTCCGCCGAAAAACAAACGAACTACG-BHQ
ITGA4 / TGCGGAGGCGTAGGGTC / CAACCGAAATTCCCCAACG / 6FAM-CCTACAACCGCGCGTAAACAAAAACG-BHQ-1
MT1A / CGTGTTTTCGTGTTATTGTGTACG / CTCGCTATCGCCTTACCTATCC / 6FAM-TCCACACCTAAATCCCTCGAACCCACT-BHQ-1
MT3 / GGTTTTAGGGTTTATGTCGAGGAGA / CCGCGCGTCCAATTACTTA / 6FAM-AAAACCCGTTCACCGCCTCCAACTACTA-BHQ-1
MYOD1 / GAGCGCGCGTAGTTAGCG / TCCGACACGCCCTTTCC / 6FAM-CTCCAACACCCGACTACTATATCCGCGAAA-BHQ-1
NEUROD1 / GTTTTTTGCGTGGGCGAAT / CCGCGCTTAACATCACTAACTAAA / 6FAM-CGCGCGACCACGACACGAAA-BHQ-1
NEUROD2 / GGTTTGGTATAGAGGTTGGTATTTCGT / ACGAACGCCGACGTCTTC / 6FAM-CGCCATACGAACCGCGAAACGAATATAA-BHQ-1
NEUROG1 / CGTGTAGCGTTCGGGTATTTGTA / CGATAATTACGAACACACTCCGAAT / 6FAM-CGATAACGACCTCCCGCGAACATAAA-BHQ-1
PENK / GGTTAATTATAAAGTGGTTTTAGTAGTCGTTAAG / CAACGTCTCTACGAAATCACGAAC / 6FAM-AACGCCTACCTCGCCGTCCCG-BHQ-1
PGR / TTATAATTCGAGGCGGTTAGTGTTT / TCGAACTTCTACTAACTCCGTACTACGA / 6FAM-ATCATCTCCGAAAATCTCAAATCCCAATAATACG-BHQ-1
PITX2 (I) / AGTTCGGTTGCGCGGTT / TACTTCCCTCCCCTACCTCGTT / 6FAM-CGACGCTCGCCCGAACGCTA-BHQ-1
PITX2 (II) / GTGGTTTCGTATTAATTAGGACGTTTC / CCGACCCCGCACTACTAATAAAT / 6FAM-ACAAAACAATCCCTCCACCGCGACT-BHQ-1
PYCARD (ASC) / TTGGAGATTTACGGCGTCG / ACCCTAATACGTAACCGCCTACAA / 6FAM-CATCTCCTACAAACCCATATCGCGCAA-BHQ-1
SFRP1 / CAACTCCCGACGAAACGAA / CGCGAGGGAGGCGATT / 6FAM-CACTCGTTACCACGTCCGTCACCG-BHQ-1
SFRP4 / TCCGCCGTCTAACACACAAA / TTCGTAATGGTCGTGGTTGGT / 6FAM-CAACGCCAACTCTCAACCTTCGAAACG-BHQ-1
SFRP5 / GAACGCCCCGACTAATCCTAA / TAGGCGGTCGGAGATTGGT / 6FAM-CTCCCACCTCGAAACTCCAACCCG-BHQ-1
SLC6A20 / AGGCGAATACGAATTGTAGCG / TAAAACGACGCGCCTAACG / 6FAM-CCGCGCACTAAAACTACCGTACCGAA-BHQ-1
SLIT2 / CAATTCTAAAAACGCACGACTCTAAA / CGGGAGATCGCGAGGAT / 6FAM-CGACCTCTCCCTCGCCCTCGACT-BHQ-1
TITF1 / CGAAATAAACCGAATCCTCCTTAA / TGTTTTGTTGTTTTAGCGTTTACGT / 6FAM-CTCGCGTTTATTTTAACCCGACGCCA-BHQ-1
TMEFF2 / CGACGAGGAGGTGTAAGGATG / CAACGCCTAACGAACGAACC / 6FAM-TATAACTTCCGCGACCGCCTCCTCCT-BHQ-1
TP73 / GGGTCGGGTAGTTCGTTTTG / CGATTTCGCTACGTCCCCT / 6FAM-AACCTCCGAACGAATACGCGAACGAA-BHQ-1
TWIST / GTAGCGCGGCGAACGT / AAACGCAACGAATCATAACCAAC / 6FAM-CCAACGCACCCAATCGCTAAACGA-BHQ-1
ZBTB16 / ATCACGACGACAACGACAACAT / TGATTTGTTAATTTCGTAGTAGAGAGGAGTT / 6FAM-CGACAATTCGCAATACCCGCTCTCA-BHQ-1
HUGO Gene Nomenclature / Alternative gene names / Chromosomal location / Amplicon start: location relative to transcription start (bp) / Amplicon end: location relative to transcription start (bp)
BCL2 / Bcl-2/B-cell CLL/lymphoma2 / 18q21.3 / 209 / 292
CACNA1G / Calcium channel, voltage-dependent, alpha 1G subunit / 17q22 / 16 / 82
CALCA / Calcitonin/Calcitonin-related polypeptide alpha; CALC1 / 11p15.2-p15.1 / -127 / -27
CDH13 / H-Cadherin/Cadherin 13, H-cadherin (heart)/T-cadherin; CDHH / 16q24.2-q24.3 / 140 / 242
CRABP1 / Cellular retinoic acid binding protein 1/RBP5, CRABP, CRABPI, CRABP-I / 15q24 / 44 / 125
CYP1B1 / Cytochrome P450, family 1, Subfamily B, polypeptide 1; GLC3A; CP1B / 2p22-p21 / -165 / -80
CYP27B1 / Cytochrome P450, family 27, subfamily B, polypeptide 1 / 12q14.1 / +404 / +482
DCC / Deleted in colorectal carcinoma; Colorectal cancer-related choromosome sequence 18 / 18q21.3 / -97 / 61
DLC1 / Deleted in liver cancer 1, HP, ARHGAP7, STARD12, FLJ21120, / 8p22-p21.3 / +221 / +144
EBF3 (DKFZ) / COE3 / 10q26 / -173 / -96
ESR1 / Estrogen receptor receptor 1, Estrogen Receptor Alpha (ESRA); NR3A1 / 6q25.1 / 14 / 114
FLJ39739 / Hypothetical FLJ39739 / 1q21.1 / -493 / -414
GABRA2 / Gamma-aminobutyric acid A receptor, alpha 2 / 4p12 / -410 / -483
GAD1 / Glutamine decarboxylase 1 (67 kda) / 2q31 / +1995 / +1915
GATA3 / GATA binding protein 3/HDR; MGC5445 / 10p15 / -478 / -399
GATA4 / GATA binding protein 4 / 8p23.1-8p22 / 393 / 466
GATA5 / GATA binding protein 5; bB379O24.1 / 20q13.33 / -156 / -240
GDNF / Glial cell derived neurotrophic factor / 5p13.1-p12 / -202 / -94
HIC1 / Hypermethylated in cancer 1; ZBTB29 / 17p13.3 / -55 / 45
HOXA1 / Homeobox A1; Homeobox 1F / 7p15.3 / 151 / 233
HOXA10 / Homeobox A10; Homeobox 1H / 7p15-p14.2 / -174 / -257
HOXA11 / Homeobox A11 / 7p15-p14.2 / -564 / -663
HOXA13 / Homeobox A13 / 7p15-p14 / 607 / 687
HOXA6 / Homeobox A6 / 7p15-p14 / -375 / -288
HOXA7 / Homeobox A7 / 7p15-p14 / -293 / -182
HOXA9 / Homeobox A9 / 7p15-p14 / -804 / -692
HOXB7 / Homeobox B7 / 17q21-q22 / -410 / -264
HOXC9 / Homeobox C9 / 12q12-q13 / -486 / -400
HOXD11 / Homeobox D11 / 2q31-q37 / -70 / 23
HOXD12 / Homeobox D12 / 2q31-q32 / -473 / -350
HOXD8 / Homeobox D8 / 2q31-q37 / -196 / -112
HOXD9 / Homeobox D9 / 2q31-q37 / -15 / 70
IGF2 / IGF2: Insulin-like growth factor 2 (somatomedin A) / 11p15.5 / -458 / -377
ITGA4 / Integrin alpha 4 (antigen CD49D, alpha-4 subunit of VLA-4 receptor); CD49D / 2q31-q32 / 796 / 866
MT1A / Metallothionein 1A/Metallothionein-I / 16q13 / -612 / -533
MT3 / Metallothionein 3 (growth inhibitory factor (neurotrophic))/GIF, GIFB, GRIF / 16q13 / +66 / -8
MYOD1 / Myogenic differentiating antigen 1; Myogenic determining factor 3/MYF3/PUM / 11p15.4 / -375 / -302
NEUROD1 / Neurogenic differentiation 1; B-cell E-box transctivator 2 (BETA2); BHF-1 / 2q32 / 35 / -47
NEUROD2 / Neurogenic differentiation 2 / 17q12 / -30 / +58
NEUROG1 / Neurogenin 1; NEOROD3; AKA / 5q23-q31 / -132 / -220
PENK / PpENK/Proenkephalin precursor / 8q23-q24 / -58 / +41
PGR / Progesterone receptor B / 11q22 / 99 / 192
PITX2 (I) / Paired-like homeodomain transcription factor 2; Pituitary homeobox 2 (PTX2) / 4q25-26 / 4818 / 4716
PITX2 (II) / Paired-like homeodomain transcription factor 2; Pituitary homeobox 2 (PTX2) / 4q25-26 / 113 / 192
PYCARD (ASC) / TMS1; CARD5; PYCARD; MGC10332 / 16p12-p11.2 / 420 / 515
SFRP1 / Secreted frizzled-related protein 1; FRP; FRP1; FrzA; SARP2 / 8p12-p11.1 / -130 / -58
SFRP4 / Secreted frizzled-related protein 4; FRPHE / 7p14-p13 / -40 / 64
SFRP5 / Secreted frizzled-related protein 5; SARP3 / 10q24.1 / -59 / 27
SLC6A20 / XT3/X transporter protein 3; Solute carrier family 6 (neurotransmitter transporter), member 20 / 3p21.3 / 326 / 192
SLIT2 / FLJ14420; SLIL3 / 4p15.2 / -390 / -489
TITF1 / Thyroid transcription factor 1; NKX2A; TEBP; BCH; TTF-1 / 14q13 / -74 / 54
TMEFF2 / Exonal sequences shared with TENB2; HPP1; TPEF / 2q32.3 / -150 / -222
TP73 / hDNp73B / 1p36.3 / -63 / 19
TWIST / Twist homolog (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) (Drosophila) / 7p21.2 / 669 / 745
ZBTB16 / Promyelocytic leukemia zinc finger protein (PLZF); ZNF145 / 11q23.2 / -32 / 55
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Case / NEUROD1 / Case / NEUROD1PMR / Expression / PMR / Expression
1 / 0.047 / 0.000 / 33 / 25.456 / 0.000
2 / 0.246 / 0.000 / 34 / 29.153 / 0.000
3 / 0.641 / 0.000 / 35 / 29.829 / 0.000
4 / 0.669 / 2.595 / 36 / 30.277 / 0.000
5 / 0.708 / 0.000 / 37 / 32.528 / 0.000
6 / 0.722 / 0.000 / 38 / 34.396 / 0.000
7 / 0.892 / 93.856 / 39 / 34.549 / 0.302
8 / 1.495 / 0.000 / 40 / 36.433 / 0.000
9 / 1.912 / 0.000 / 41 / 36.685 / 0.000
10 / 3.205 / 0.000 / 42 / 39.516 / 0.000
11 / 3.628 / 0.000 / 43 / 46.768 / 0.000
12 / 4.412 / 0.000 / 44 / 46.908 / 0.000
13 / 4.898 / 0.000 / 45 / 47.793 / 0.000
14 / 5.067 / 0.000 / 46 / 52.011 / 0.000
15 / 6.855 / 0.000 / 47 / 56.516 / 1.064
16 / 6.875 / 0.000 / 48 / 59.441 / 0.416
17 / 6.889 / 0.000 / 49 / 61.509 / 0.000
18 / 7.02 / 0.000 / 50 / 63.278 / 0.000
19 / 7.234 / 0.000 / 51 / 71.241 / 2.882
20 / 9.178 / 0.000 / 52 / 75.363 / 0.000
21 / 9.866 / 0.000 / 53 / 75.382 / 0.000
22 / 10.85 / 0.000 / 54 / 75.608 / 1.959
23 / 12.713 / 0.000 / 55 / 79.196 / 0.000
24 / 12.82 / 0.000 / 56 / 79.849 / 0.000
25 / 13.714 / 0.000 / 57 / 84.717 / 0.000
26 / 15.965 / 0.000 / 58 / 85.034 / 0.000
27 / 16.285 / 0.000 / 59 / 86.433 / 0.000
28 / 17.025 / 0.849 / 60 / 86.963 / 0.000
29 / 18.225 / 0.000 / 61 / 95.934 / 0.000
30 / 19.358 / 0.000 / 62 / 202.137 / 0.000
31 / 23.485 / 1.125 / 63 / 482.26 / 0.000
32 / 23.731 / 0.000
Supplemental Table S3. NEUROD1 methylation and mRNA expression in 63 breast cancer specimens. PMR=Percentage of Methylated Reference
Non Cancer Specific / pGenes / value
SFRP1 / 0.063
NEUROD2 / 0.099
PGR / 0.109
GATA5 / 0.114
ESR1 / 0.144
DLC1 / 0.180
GATA3 / 0.180
GDNF / 0.180
MYOD1 / 0.203
CACNA1G / 0.285
CDH13 / 0.310
CYP1B1 / 0.317
SLC6A20 / 0.317
EBF3 (DKFZ) / 0.398
ZBTB16 / 0.401
MT3 / 0.424
HOXA11 / 0.470
HOXA6 / 0.484
GABRA2 / 0.500
SFRP5 / 0.534
HOXA10 / 0.551
CYP27B1 / 0.594
DCC / 0.655
HOXC9 / 0.655
IGF2 / 0.655
NEUROG1 / 0.655
TITF1 / 0.655
CALCA / 0.674
FLJ39739 / 0.674
HOXB7 / 0.730
SLIT2 / 0.790
ITGA4 / 0.893
PITX2 (I) / 0.893
TWIST / 0.893
SFRP4 / 0.917
HOXA9 / 0.925
HOXD8 / 0.972
BCL2 / 1.000
PYCARD / 1.000
TP73 / 1.000
Supplemental Table S4. Comparison of DNA methylation changes in non-cancer specific genes between tumour taken from the centre (TUC) and tumour taken from the periphery (TUP) with non-parametric paired test analysis.
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