Bin Liu, Lei Yang, Binfang Huang, Mei Cheng, Hui Wang, Yinyan Li, Dongsheng Huang, Jian Zheng,
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1 The American Journal of Human Genetics, Volume 91 Supplemental Data A Functional Copy-Number Variation in MAPKAPK2 Predicts Risk and Survival of Lung Cancer Bin Liu, Lei Yang, Binfang Huang, Mei Cheng, Hui Wang, Yinyan Li, Dongsheng Huang, Jian Zheng, Qingchu Li, Xin Zhang, Weidong Ji, Yifeng Zhou, and Jiachun Lu Figure S1. The Chromosomal Location of MAPKAPK2 with CNVs Information and Genotyping of g.cnv (A) Schematic of MAPKAPK2 in Chr.1q32 and the three identified CNVs.
2 (B) g.cnv genotyping by the Taqman real-time PCR: in 10µl reaction systems, triplicate 10-ng DNA samples were mixed with TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA, USA), the control RNase P probe (VIC labeled, Applied Biosystems) and the experimental probe (FAM labeled, cat# Hs Applied Biosystems), then the mixture were run on an ABI 7900HT fast real-time PCR System (Applied Biosystems) and the genotypes were automatically determined by software CopyCaller 1.0 (Applied BioSystems). The mixture without genomic DNA was defined as zero copy number and 3 samples with two copies of g.cnv locus found by Affymetrix Genome-Wide Human SNP Array 6.0 were used as standard sample. Columns, mean from three independent experiments; bars, SD. (C) g.cnv genotyping by the Accucopy assay with a patented Multiplex AccuCopy TM Kit (Genesky Bio-Tech Co., Ltd., Shanghai, China).
3 Figure S2. MAPKAPK2 g.cnv Calculation with Affymetrix Genome-wide Human SNP Array 6.0 (A) 3-copy genotype. (B) 4- copy genotype.
4 Figure S3. Stratification Analysis of the g.cnv Genotypes and Lung Cancer Risk P value for the homogeneity test in each stratum was tested by Breslow-Day Test. A multiplicative interaction model was applied for the interaction analysis.
5 Figure S4. Stratification Analysis of the g.cnv Genotypes and Lung Cancer Survival A multiplicative interaction model was supplied for the interaction analysis.
6 Figure S5. MAPKAPK2 Protein Expressions in Lung Cancer Tissues In Situ by Immunohistochemistry The MAPKAPK2 cytoplasmic expression was scored to the sum of the intensity and percentage and defined as follows: 0-2, negative or weak; 3-4, moderate; and 5-6, strong by using the Image Pro Plus software. A, 2-copy genotype. B, 3-copy genotype. C, 4-copy genotype. D, MAPKAPK2 expression in situ by g.cnv genotypes. Columns, mean from three independent experiments; bars, SD. The copy number of g.cnv was CNV in blood DNA. The 4 copies of g.cnv had significantly higher MAPKAPK2 expressions (both intensity and positive percentage) than that of 2 or 3 copies as ANOVA test shown.
7 Figure S6. The Relationship between g.cnv in Peripheral-Blood and Tumor Tissues The 4 copies genotype might cause a more copy number variation in somatic genome.
8 Table S1. Frequency Distributions of Selected Variables in Individuals with Lung Cancer and Cancer-free Controls Variables Discovery set (Southern Chinese) Validation set I (Eastern Chinese) Validation set II (Southern Chinese) Case (n=1056) Control (n=1056) P a Case (n=503) Control (n=623) P a Case (n=773) Control (n=778) Age (years) (50.8) 534(50.6) (54.3) 343(55.1) (56.7) 447(57.5) > (49.2) 522(49.4) 230(45.7) 280(44.9) 335(43.3) 331(42.5) Sex Male 746(70.6) 746(70.6) (66.6) 433(70.4) (70.5) 547(70.3) Female 310(29.4) 310(29.4) 158(31.4) 184(29.6) 228(29.5) 231(29.7) Family history of cancer Yes 104(9.9) 103(9.8) (5.0) 44(7.1) (4.1) 47(6.1) No 952(90.1) 953(90.2) 478(95.0) 579(92.9) 741(95.9) 730(93.9) Family history of lung cancer Yes 42(4.0) 30(2.8) (2.0) 13(2.1) (3.5) 14(1.8) No 1014(96.0) 1026(97.2) 493(98.0) 610(97.9) 746(96.5) 763(98.2) Pre-existing COPD Yes 148(14.0) 113(10.7) (13.7) 56(9.0) (8.7) 43(5.5) No 908(86.0) 943(89.3) 434(86.3) 567(91.0) 706(91.3) 735(94.5) Smoking status Current 394(37.3) 366(34.6) (23.5) 168(26.6) (24.2) 219(28.1) Former 207(19.3) 176(16.8) 105(20.9) 57(9.2) 197(25.5) 80(10.3) Never 455(43.1) 514(48.7) 280(55.6) 400(64.2) 389(50.3) 479(61.6) Pack-years smoked (43.5) 314(29.7) (32.8) 165(26.5) (37.7) 218(28.0) <20 142(13.4) 228(21.6) 58(11.5) 58(9.3) 93(12.0) 81(10.4) P a
9 0 455(43.1) 514(48.7) 280(55.7) 400(64.2) 389(50.3) 479(61.6) Drinking status Current 165(15.6) 186(17.6) (7.2) 75(12.0) (10.4) 106(13.6) Former 64(6.1) 41(3.9) 28(5.6) 40(6.4) 75(9.7) 59(7.6) Never 827(78.3) 829(78.5) 439(87.2) 508(81.6) 618(79.9) 613(78.8) Histological types Adenocarcinoma 384(36.4) 231(45.9) 330(42.7) Squamous cell carcinoma 369(34.9) 158(31.4) 238(30.8) Large cell carcinoma 43(4.1) 23(4.6) 35(4.5) Small cell lung cancer 128(12.1) 65(12.9) 92(11.9) Other carcinomas b 132(12.5) 26(5.2) 78(10.1) Stages I 154(14.6) 46(9.2) 98(12.7) II 94(8.9) 53(10.5) 83(10.8) III 333(31.5) 157(31.2) 214(27.7) IV 475(45.0) 247(49.1) 378(48.8) a P values for a χ 2 test. b Mixed-cell or undifferentiated carcinoma.
10 Table S2. Analysis of the Effects of Lung Cancer-Affected Individuals Characteristics and Clinical Features on Lung Cancer Survival Variables Discovery set (Southern Chinese) Case No. of death Validation set I (Eastern Chinese) Case No. of death Validation set II (Southern Chinese) Case No. of MST (months) Log-rank P value Total Age (years) death Cox model Crude HR (95%CI) Cox model Adjust a HR (95%CI) (44.9) (51.0) (52.6) (ref.) 1.00 (ref.) > (50.1) (49.0) (47.4) ( ) 1.24 ( ) Sex Male 366 (71.8) (67.6) (69.2) (ref.) 1.00 (ref.) Female 144 (28.2) (32.4) (30.8) ( ) 0.91 ( ) Family history of cancer No 461 (90.4) (96.3) (96.1) (ref.) 1.00 (ref.) Yes 49 (9.6) (3.7) 6 13 (3.9) ( ) 0.81 ( ) Family history of lung cancer No 489 (95.9) (98.6) (97.3) (ref.) 1.00 (ref.) Yes 21 (4.1) 16 4 (1.4) 2 9 (2.7) ( ) 0.89 ( ) Pre-existing COPD No 72(14.1) (85.1) (90.6) (ref.) 1.00 (ref.) Yes 438(85.9) (14.9) 38 31(9.4) ( ) 1.15 ( ) Smoking status Never 211 (41.4) (55.7) (54.7) (ref.) 1.00 (ref.) Ever 299 (58.6) (42.3) (45.3) ( ) 1.19 ( ) Pack-years smoked (43.3) (56.1) (54.7) (ref.) 1.00 (ref.) <20 59 (11.6) (8.1) (7.8) ( ) 1.06 ( )
11 (47.1) (35.8) (34.5) ( ) 1.09 ( ) Drinking status Never 401 (78.6) (88.5) (87.0) (ref.) 1.00 (ref.) Ever 109 (21.4) (11.5) (13.0) ( ) 1.08 ( ) Surgery No 334 (65.5) (54.7) (57.7) (ref.) 1.00 (ref.) Yes 176 (34.5) (45.3) (42.3) ( ) 0.57( ) Chemotherapy No 203 (39.8) (28.7) (27.5) (ref.) 1.00 (ref.) Yes 307 (60.2) (71.3) (72.5) ( ) 0.77( ) Radiotherapy No 274 (53.7) (39.9) (46.2) (ref.) 1.00 (ref.) Yes 236 (46.3) (60.1) (53.8) ( ) 0.86( ) Stages I+ II 96 (18.8) (19.6) (22.7) (ref.) 1.00 (ref.) III 169 (33.1) (31.8) (49.0) ( ) 1.76 ( ) IV 245 (48.1) (48.6) (28.3) ( ) 1.38 ( ) Histological types Adenocarcinoma 179 (35.1) (44.6) (44.7) (ref.) 1.00 (ref.) Squamous cell carcinoma 164 (32.2) (32.1) (31.4) ( ) 0.92 ( ) Large cell carcinoma 24 (4.7) (5.7) (5.8) ( ) 0.99 ( ) Small cell lung cancer 62 (12.1) (13.2) (12.7) ( ) 1.04 ( ) Other b 81 (15.9) (4.4) 7 18 (5.4) ( ) 0.96 ( ) Abbrevations: MST, median survival time; HR, hazard ratio; a Adjusted in a multivariate Cox regression model that included age, sex, pre-existing COPD, smoking status, stages, surgery, chemotherapy, and radiotherapy status. b Mixed-cell or undifferentiated carcinoma.
12 Table S3. Multivariate Cox Regression Analysis of Surrounding Factors and Lung Cancer Survival Factors HR (95%CI) a COX mode P value age 1.25 ( ) sex 0.92 ( ) Pre-existing COPD 1.12 ( ) Smoking status 1.20 ( ) Surgery 0.57 ( ) Chemotherapy 0.78 ( ) Radiotherapy 0.85 ( ) Stages 1.26 ( ) MAPKAPK2 g.cnv a 1.49 ( ) a g.cnv-30450: 4 copies vs. 2/3 copies. Table S4. The Primers Used for SYBR-Green Real-Time PCR Gene Primer MAPKAPK2 5 -CAGCAGTTCCCGCAGTTC-3 (forward) 5 -CGAATTTCTCCTGGGTCCTC-3 (reverse) β-actin 5 -GGCGGCACCACCATGTACCCT-3 (forward) 5 -AGGGGCCGGACTCGTCATACT-3 (reverse)
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