Supplementary Tables and Figures

SUPPLEMENTARY TABLES AND FIGURES

Table S1. CRC and extracolonic cancers in the families included in the linkage analysis.

FAMILY / NUMBER AND TYPE OF CANCER
93C / 6 CRC - 1 Pancreas – 1 Digestive
95C / 7 CRC - 1 Gastric– 1 Pancreas – 3 Polyps
174C / 7 CRC - 3 Lung – 2 Breast – 1 Digestive – 1 Lymphoma NH
177C / 3 CRC - 1 Peritoneum
191C / 4 CRC - 1 Polyp
192C / 4 CRC - 2 Lung – 1 Breast – 1 Leukemia
200C / 5 CRC
263C / 4 CRC - 2 Ovary – 1 Lung – 2 Lymphoma – 1 Prostate
266C / 4 CRC - 1 CNS
274C / 5 CRC
381C / 4 CRC - 1 Breast – 4 Polyps
474C / 4 CRC - 1 CNS
558C / 5 CRC - 4 Polyps
603C / 4 CCR - 1 Skin – 1 Polyp
874C / 4 CRC - 2 Endometrial
1081C / 2 CRC - 1 Ovary – 1 Polyp
1084C / 2 CRC - 1 Prostate – 1 Pancreas – 1 Polyp
1111C / 4 CRC - 1 Prostate – 1 Polyp
1113C / 3 CRC - 2 Breast
1069C / 3 CRC - 3 Breast – 2 Gastric – 1 Skin
1089C / 2 CRC - 1 Uterus
1110C / 5 CRC - 1 Lung – 1 Skin – 1 Prostate – 1 Gastric – 1 Tongue – 1 Polyp

Table S2. Characteristics of the microsatellites used in the fine-mapping analysis

Marker / cM / Heterozygosity / Size (range) / Position / Forward Primer / Reverse Primer
D12S92 / 84.2 / 0.75 / 188-204 / Ch 12: 74133886-74134061 / TGCCCAAGTTGCAGATG / GTCAGGATTCCCCAGAAAA
D12S1660 / 84.7 / 0.89 / 196-226 / Ch 12: 76462424-76462624 / GTTTGACACAAGTACACCAAGTCAC / AAGTACCAGCCTATTTTTTTGGACC
D12S1684 / 87.6 / 0.90 / 226-256 / Ch 12: 77263237-77263478 / GCCTCAGTTATGAGCCAATAAATTC / GTAAGCCTGCTAACATTTGTGGAC
D12S326 / 87.6 / 0.79 / 237-261 / Ch 12: 77973840-77974092 / CCCAGCAGTGCTAGTGTTGA / GGGCTAGGGTGGAGAATCAA
D12S1708 / 92.6 / 0.72 / 167-175 / Ch 12: 82,405,262-82,405,430 / GGGAACTTATGTCAAGGCTAGGA / GATCTAGTGCTCAAGAGGTTTTCAA
D12S1667 / 94.2 / 0.77 / 94-112 / Ch 12: 83,730,951-83,731,054 / CCCAGGATATGTAAGTAAATCCAA / TCCCCATTTTTTAATCAGGT
D12S311 / 96.5 / 0.57 / 199-207 / Ch 12: 92,707,048-92,707,252 / CCAAACATTAACTGTTCCC / GTGCCCTGAGCAACTG
D12S1345 / 97.6 / 0.75 / 214-236 / Ch 12: 93,070,072-93,070,305 / GCTCTGAAAAGCACATGACC / AGCTACTCTAAGGGAAAAGACAG

Table S3. Antibodies used in the tissue microarray.

PROTEIN / CLONE / SUPPLIER
Mlh1 / ES05 / Dako
Msh2 / FE11 / Oncogene
Msh6 / 44 / BD Transduction Lab
Hes 1 / Polyclonal / Santa Cruz
SMAD4 / B8 / Santa Cruz
E-CADHERIN/CDH1 / 4A2C7 / Zymed
PTEN / 138G6 / Cell Signaling
PI3KCA / C73F8 Rabbit Monoclonal / Cell Signaling
MUC-1 / E29 / Dako
Bcl-2 / 124 / DAKO
CDk2 Ab-2 / 8D4 / Neo Markers
Ciclina D1 / SP4 Rabbit / Dako
p21 (WAF1) / EA10 / CALBIOCHEM
p53 / DO-7 / Dako
RB-P / G3-245 / BD PharMingen
COX2/ PTGS2 / SP21 Rabbit Monoclonal / Neomarkers
EGFR / EGFR.113 / Novocastra
B-CATHENIN / Β-Catenin-1 / Dako
P16/ CDKN2A / E6H4 / Mtm
hTERT / 44F12 / Novocastra
NTS / NA 1230 / Enzo
NTSR1 / Rabbit Polyclonal / GenWay

.

Table S4. Characteristics of the candidate regions identified by nonparametric linkage (NPL) analysis.

REGION / FROM
(Position bp) / TO
(Position bp) / NPL-SCORE / pVALUE / Size (bp)
2p24.3 / 14751347 / 15625817 / 2.13 / 0.00209 / 874470
4q13.1 / 61686118 / 64954857 / 2.43 / 0.005197 / 3268739
4q31.21 / 144896743 / 145879472 / 2.35 / 0.007576 / 982729
12q21.2 – 12q21.31 / 85789974 / 96450337 / 2.47 / 0.002389 / 10660363

Table S5. Genes contained in the region 12q21.2 – 12q21.31.

ENSEMBL ID / GENE / LOCATION
ENSG00000201942 / 5S_rRNA / q21.2
ENSG00000244015 / AC079595.1 / q21.31
ENSG00000243714 / AC027288.1 / q21.2
ENSG00000238769 / snoU13 / q21.2
ENSG00000221148 / SNORA3 / q21.31
ENSG00000238842 / U7 / q21.31
ENSG00000252220 / U6 / q21.31
ENSG00000249929 / AC128707.1 / q21.2
ENSG00000213272 / RP11-613M5.1 / q21.2
ENSG00000243071 / RP11-613M5.2 / q21.2
ENSG00000241045 / RP11-613M5.3 / q21.2
ENSG00000213271 / AC068993.1 / q21.2
ENSG00000239473 / RP1-170B16.1 / q21.31
ENSG00000230291 / RP11-244J10.1 / q21.31
ENSG00000213270 / RP11-87P13.1 / q21.31
ENSG00000179941 / BBS10 / q21.2
ENSG00000091039 / OSBPL8 / q21.2
ENSG00000186908 / ZDHHC17 / q21.2
ENSG00000175183 / CSRP2 / q21.2
ENSG00000165891 / E2F7 / q21.2
ENSG00000231121 / AC073528.1 / q21.2
ENSG00000067798 / NAV3 / q21.2
ENSG00000067715 / SYT1 / q21.2
ENSG00000235796 / AC027288.2 / q21.2
ENSG00000177425 / PAWR / q21.2
ENSG00000058272 / PPP1R12A / q21.31
ENSG00000165899 / C12orf64 / q21.31
ENSG00000139304 / PTPRQ / q21.31
ENSG00000111046 / MYF6 / q21.31
ENSG00000111049 / MYF5 / q21.31
ENSG00000111052 / LIN7A / q21.31
ENSG00000111058 / ACSS3 / q21.31
ENSG00000139220 / PPFIA2 / q21.31
ENSG00000133773 / CCDC59 / q21.31
ENSG00000127720 / C12orf26 / q21.31
ENSG00000179104 / TMTC2 / q21.31
ENSG00000072041 / SLC6A15 / q21.31
ENSG00000231738 / TSPAN19 / q21.31
ENSG00000133640 / LRRIQ1 / q21.31
ENSG00000180318 / ALX1 / q21.31
ENSG00000198774 / RASSF9 / q21.31
ENSG00000133636 / NTS / q21.31
ENSG00000182050 / MGAT4C / q21.31
ENSG00000238890 / AC078917.1 / q21.2
ENSG00000221788 / hsa-mir-1252 / q21.2
ENSG00000207763 / hsa-mir-617 / q21.31
ENSG00000208022 / hsa-mir-618 / q21.31
ENSG00000221121 / AC083811.1 / q21.31
ENSG00000212029 / AC079595.2 / q21.31
ENSG00000223273 / 7SK / q21.2
ENSG00000222880 / 7SK / q21.31

Table S6. Proteins with different immunohistochemical expression in groups A and B.

MARKER / P-VALUE
MLH1 / <0.001
MSH2 / <0.001
MSH6 / <0.001
E-CADHERIN / <0.001
CDK2 / <0.001
CICLINA D1 / <0.001
RB-P / <0.001
P21 / 0.001
BCL2 / 0.001
EGFR / 0.002
PI3KCA / 0.005
COX2 / 0.018
SMAD4 / 0.020
BETA CATENIN / 0.024
P53 / 0.035

Table S7. Proteins with different immunohistochemical expression between sub-clusters A1 and A2.

MARKER / P-VALUE
MLH1 / <0.001
MSH2 / <0.001
MSH6 / <0.001
P53 / <0.001
E-CADHERIN / <0.001
SMAD4 / <0.001
PI3KCA / 0.001
P21 / 0.002
MUC1 / 0.003
EGFR / 0.003
hTERT / 0.010
NTS / 0.011
CDK2 / 0.015
RBP / 0.015
CYCLIN D1 / 0.024

Table S8. SNPs associated with CRC risk in GWAS studies.

SNP / REGION / GEN / CHROMOSOME / OR / P-VALUE / STUDY
rs11903757 / intronic / NABP1 / Chr2 / 1.15 (1.09–1.22) / 1.38×10-6 / Peters et al. 20131
rs10911251 / intronic / LAMC1 / Chr1 / 1.10 (1.06–1.14) / 1.34×10-6
rs3217810 / intronic / CCND2 / Chr12 / 1.19 (1.11–1.28) / 3.40×10-7
rs3217901 / intronic / CCND2 / Chr12 / 0.41 (0.43–0.39) / 1.71×10-6
rs59336 / intronic / TBX3 / Chr12 / 1.10 (1.06–1.14) / 7.64×10-7
rs4779584 / intergenic / SCG5-GREM1 / Chr15 / 1.18 (1.11-1.24) / 2.00×10-8 / Peters et al.
20112
rs4939827 / Intronic / SMAD7 / Chr18 / 1.14 (1.08-1.18) / 1.00×10-7
rs16892766 / intergenic / EIF3H / Chr8 / 1.24 (1.14-134) / 4.00×10-7
rs7315438 / intergenic / med13l / Chr12 / 1.11 (1.06-1.15) / 6.00×10-6
rs7758229 / Intronic / slo22a3 / Chr6 / 1.28 (1.18-1.39) / 8.00×10-9 / Cui et al. 20113
rs6983267 / Intergenic / pou5f1b / Chr8 / 1.18 (1.11-1.25) / 2.00×10-8
rs11169552 / intergenic / atf1 / Chr12 / 1.09 (1.05-1.11) / 2.00×10-10 / Houlston et al. 20104
rs4925386 / Intronic / lama5 / Chr20 / 1.08 (1.05-1.10) / 2.00×10-10
rs6691170 / Intergenic / dusp10 / Chr1 / 1.06 (1-03-1.09) / 1.00×10-19
rs6687758 / intergenic / dusp10 / Chr1 / 1.09 (1.06-1.12) / 2.00×10-9
rs10936599 / cds-symon / MYNN / Chr3 / 1.04 (1.04-1.10) / 3.00×10-8
rs961253 / Intergenic / bmp2 / Chr20 / 1.12 (1.08-1.16) / 2.00×10-10 / Houlston et al. 20085
rs4444235 / Intergenic / bmp4 / Chr14 / 1.11 (1.08-1.15) / 8.00×10-10
rs10411210 / Intronic / rhpn2 / Chr19 / 1.15 (1.10-1.20) / 5.00×10-9
rs9929218 / Intronic / cdh1 / Chr16 / 1.1 (1.06_1.12) / 1.00×10-8
rs7014346 / Intergenic / POU5FIP1 / Chr8 / 1.19 (1.15-1.23) / 9.00×10-26 / Tenesa et al. 20086
rs3802842 / Intronic / c11orf93 / Chr11 / 1.11 (1.08-1.15) / 6.00×10-10
rs10795668 / Intergenic / SFTA1P / Chr10 / 1.12 (1.10-1.16) / 3.00×10-13
rs16892766- / intergenic / TRPS1
EIF3H / Chr8 / 1.27 (1.20-1.34) / 1.00×10-18 / Tomlinson et al. 20087
rs10795668- / intergenic / KRT8P16
TCEB1P3 / Chr10 / 1.12 (1.10-1.16) / 1.00×10-13
rs6983267 / intergenic / SRRM1P1
POU5F1B / Chr24 / 1.24 (1.17-1.33) / 1.00×10-11
rs4779584 / intergenic / SCG5
GREM1 / Chr15 / 1.23 (1.14-1.34) / 1.00×10-7
rs4939827 / intronic / SMAD7 / Chr18 / 1.18 (1.10-1.25) / 1.00×10-6
rs10505477 / Intergenic / POU5F1B / Chr8 / 1.17 (1.12-1.23) / 3.00×10-11 / Zanke et al. 20078
rs4939827 / Intronic / SMAD7 / Chr18 / 1.16 (1.09-1.27) / 1.00×10-12 / Broderick et al. 20079
rs6983267 / Intergenic / SRRM1P1
POU5F1B / Chr8 / 1.27 (1.16-1.39) / 1.00×10-14 / Tomlinson et al. 200710

1. Peters U, Jiao S, Schumacher FR, Hutter CM, Aragaki AK, Baron JA, Berndt SI, Bezieau S, Brenner H, Butterbach K, Caan BJ, Campbell PT, Carlson CS, Casey G, Chan AT, Chang-Claude J, Chanock SJ, Chen LS, Coetzee GA, Coetzee SG, Conti DV, Curtis KR, Duggan D, Edwards T, Fuchs CS, Gallinger S, Giovannucci EL, Gogarten SM, Gruber SB, Haile RW, Harrison TA, Hayes RB, Henderson BE, Hoffmeister M, Hopper JL, Hudson TJ, Hunter DJ, Jackson RD, Jee SH, Jenkins MA, Jia WH, Kolonel LN, Kooperberg C, Kury S, Lacroix AZ, Laurie CC, Laurie CA, Le Marchand L, Lemire M, Levine D, Lindor NM, Liu Y, Ma J, Makar KW, Matsuo K, Newcomb PA, Potter JD, Prentice RL, Qu C, Rohan T, Rosse SA, Schoen RE, Seminara D, Shrubsole M, Shu XO, Slattery ML, Taverna D, Thibodeau SN, Ulrich CM, White E, Xiang Y, Zanke BW, Zeng YX, Zhang B, Zheng W, Hsu L. Identification of Genetic Susceptibility Loci for Colorectal Tumors in a Genome-Wide Meta-analysis. Gastroenterology 2013.

2. Peters U, Jiao S, Schumacher FR, Hutter CM, Aragaki AK, Baron JA, Berndt SI, Bezieau S, Brenner H, Butterbach K, Caan BJ, Campbell PT, Carlson CS, Casey G, Chan AT, Chang-Claude J, Chanock SJ, Chen LS, Coetzee GA, Coetzee SG, Conti DV, Curtis KR, Duggan D, Edwards T, Fuchs CS, Gallinger S, Giovannucci EL, Gogarten SM, Gruber SB, Haile RW, Harrison TA, Hayes RB, Henderson BE, Hoffmeister M, Hopper JL, Hudson TJ, Hunter DJ, Jackson RD, Jee SH, Jenkins MA, Jia WH, Kolonel LN, Kooperberg C, Kury S, Lacroix AZ, Laurie CC, Laurie CA, Le Marchand L, Lemire M, Levine D, Lindor NM, Liu Y, Ma J, Makar KW, Matsuo K, Newcomb PA, Potter JD, Prentice RL, Qu C, Rohan T, Rosse SA, Schoen RE, Seminara D, Shrubsole M, Shu XO, Slattery ML, Taverna D, Thibodeau SN, Ulrich CM, White E, Xiang Y, Zanke BW, Zeng YX, Zhang B, Zheng W, Hsu L. Identification of Genetic Susceptibility Loci for Colorectal Tumors in a Genome-wide Meta-analysis. Gastroenterology 2011.

3. Cui R, Okada Y, Jang SG, Ku JL, Park JG, Kamatani Y, Hosono N, Tsunoda T, Kumar V, Tanikawa C, Kamatani N, Yamada R, Kubo M, Nakamura Y, Matsuda K. Common variant in 6q26-q27 is associated with distal colon cancer in an Asian population. Gut 2011;60:799-805.

4. Houlston RS, Cheadle J, Dobbins SE, Tenesa A, Jones AM, Howarth K, Spain SL, Broderick P, Domingo E, Farrington S, Prendergast JG, Pittman AM, Theodoratou E, Smith CG, Olver B, Walther A, Barnetson RA, Churchman M, Jaeger EE, Penegar S, Barclay E, Martin L, Gorman M, Mager R, Johnstone E, Midgley R, Niittymaki I, Tuupanen S, Colley J, Idziaszczyk S, Thomas HJ, Lucassen AM, Evans DG, Maher ER, Maughan T, Dimas A, Dermitzakis E, Cazier JB, Aaltonen LA, Pharoah P, Kerr DJ, Carvajal-Carmona LG, Campbell H, Dunlop MG, Tomlinson IP. Meta-analysis of three genome-wide association studies identifies susceptibility loci for colorectal cancer at 1q41, 3q26.2, 12q13.13 and 20q13.33. Nature genetics 2010;42:973-7.

5. Houlston RS, Webb E, Broderick P, Pittman AM, Di Bernardo MC, Lubbe S, Chandler I, Vijayakrishnan J, Sullivan K, Penegar S, Carvajal-Carmona L, Howarth K, Jaeger E, Spain SL, Walther A, Barclay E, Martin L, Gorman M, Domingo E, Teixeira AS, Kerr D, Cazier JB, Niittymaki I, Tuupanen S, Karhu A, Aaltonen LA, Tomlinson IP, Farrington SM, Tenesa A, Prendergast JG, Barnetson RA, Cetnarskyj R, Porteous ME, Pharoah PD, Koessler T, Hampe J, Buch S, Schafmayer C, Tepel J, Schreiber S, Volzke H, Chang-Claude J, Hoffmeister M, Brenner H, Zanke BW, Montpetit A, Hudson TJ, Gallinger S, Campbell H, Dunlop MG. Meta-analysis of genome-wide association data identifies four new susceptibility loci for colorectal cancer. Nature genetics 2008;40:1426-35.

6. Tenesa A, Farrington SM, Prendergast JG, Porteous ME, Walker M, Haq N, Barnetson RA, Theodoratou E, Cetnarskyj R, Cartwright N, Semple C, Clark AJ, Reid FJ, Smith LA, Kavoussanakis K, Koessler T, Pharoah PD, Buch S, Schafmayer C, Tepel J, Schreiber S, Volzke H, Schmidt CO, Hampe J, Chang-Claude J, Hoffmeister M, Brenner H, Wilkening S, Canzian F, Capella G, Moreno V, Deary IJ, Starr JM, Tomlinson IP, Kemp Z, Howarth K, Carvajal-Carmona L, Webb E, Broderick P, Vijayakrishnan J, Houlston RS, Rennert G, Ballinger D, Rozek L, Gruber SB, Matsuda K, Kidokoro T, Nakamura Y, Zanke BW, Greenwood CM, Rangrej J, Kustra R, Montpetit A, Hudson TJ, Gallinger S, Campbell H, Dunlop MG. Genome-wide association scan identifies a colorectal cancer susceptibility locus on 11q23 and replicates risk loci at 8q24 and 18q21. Nature genetics 2008;40:631-7.

7. Tomlinson IP, Webb E, Carvajal-Carmona L, Broderick P, Howarth K, Pittman AM, Spain S, Lubbe S, Walther A, Sullivan K, Jaeger E, Fielding S, Rowan A, Vijayakrishnan J, Domingo E, Chandler I, Kemp Z, Qureshi M, Farrington SM, Tenesa A, Prendergast JG, Barnetson RA, Penegar S, Barclay E, Wood W, Martin L, Gorman M, Thomas H, Peto J, Bishop DT, Gray R, Maher ER, Lucassen A, Kerr D, Evans DG, Schafmayer C, Buch S, Volzke H, Hampe J, Schreiber S, John U, Koessler T, Pharoah P, van Wezel T, Morreau H, Wijnen JT, Hopper JL, Southey MC, Giles GG, Severi G, Castellvi-Bel S, Ruiz-Ponte C, Carracedo A, Castells A, Forsti A, Hemminki K, Vodicka P, Naccarati A, Lipton L, Ho JW, Cheng KK, Sham PC, Luk J, Agundez JA, Ladero JM, de la Hoya M, Caldes T, Niittymaki I, Tuupanen S, Karhu A, Aaltonen L, Cazier JB, Campbell H, Dunlop MG, Houlston RS. A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3. Nature genetics 2008;40:623-30.