A genome-wide association study identifies two new cervical cancer susceptibility loci at 4q12 and 17q12. Supplementary Materials

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1 A genome-wide association study identifies two new cervical cancer susceptibility loci at 4q12 and 17q12 Supplementary Materials Yongyong Shi 1-4,42, Li Li 5,42, Zhibin Hu 6,7,42, Shuang Li 1,42, Shixuan Wang 1,42, Jihong Liu 8,42, Chen Wu 9,42, Lin He 2-4, Jianfeng Zhou 10, Zhiqiang Li 2-4, Ting Hu 1, Yile Chen 11, Yao Jia 1, Shaoshuai Wang 1, Li Wu 11, Xiaodong Cheng 12, Zhijun Yang 5, Ru Yang 1, Xiong Li 1, Kecheng Huang 1, Qinghua Zhang 1, Hang Zhou 1, Fangxu Tang 1, Zhilan Chen 1, Jian Shen 1, Jie Jiang 6,7, Hu Ding 10, Hui Xing 13, Shulan Zhang 14, Pengpeng Qu 15, Xiaojie Song 16, Zhongqiu Lin 17, Dongrui Deng 1, Ling Xi 1, Weiguo Lv 12, Xiaobing Han 18, Guangshi Tao 19, Lixing Yan 20, Zhedong Han 20, Zhuang Li 5, Xiaoping Miao 21, Shandong Pan 6,7, Yuanming Shen 12, Hui Wang 1, Dan Liu 1, Ee Gong 1, Zheng Li 8, Limei Zhou 15, Xiaomei Luan 14, Chuping Wang 22, Qian Song 23, Sufang Wu 24, Hongbin Xu 25, Jiawei Shen 2-4, Fulin Qiang 26, Gang Ma 27, Li Liu 26, Xiaojun Chen 26, Jibin Liu 26, Jiangping Wu 28, Yan Shen 28, Yang Wen 6,7, Minjie Chu 6,7, Jiang Yu 29, Xiaoxia Hu 30, Yujuan Fan 31, Hongying He 32, Yanming Jiang 33, Zhiying Lei 34, Cui Liu 35, Jianhua Chen 2, 36, Yuan Zhang 37, Cunjian Yi 38, Shuangyun Chen 39, Wenjin Li 2, Daowen Wang 10, Zehua Wang 37, Wen Di 40, Keng Shen 41, Dongxin Lin 9, Hongbing Shen 6,7, Youji Feng 24, Xing Xie 12, Ding Ma 1 1

2 Index Supplementary Figure 1. The principal components analysis (PCA) of all initial GWAS samples (1,364 cases and 3,028 controls) and HapMap 270 individuals (JPT+CHB, CEU and YRI) implemented in the software package EIGENSTRAT. Supplementary Figure 2. Plots of the first two components derived from a principle component analysis implemented in the software package EIGENSTRAT. Supplementary Figure 3. Quantile-Quantile plots of observed P values in log 10 scale. Supplementary Figure 4. Scatter plot of P values in log 10 scale in the Initial discovery set. Supplementary Figure 5. Regional plot of the four identified marker SNPs, including rs at 4q12 (a), rs at 17q12 (b), rs and rs at 6p21.32 (c-d). Results ( log 10 P) are shown for SNPs in the region flanking 150 kb on either side of the marker SNPs in the Initial discovery set. Supplementary Table 1. Associations results of 22 SNPs in the first stage of GWAS. Supplementary Table 2. Associations results of 22 SNPs in the Follow-up 1. Supplementary Table 3. Associations results of 13 SNPs in the Follow-up 2. Supplementary Table 4. Association results of 13 SNPs in the Matched discovery study, case-control validation and the combined samples. 2

3 Supplementary Table 5. Association results of 13 SNPs in the Stringent-matched discovery study and the combined samples. Supplementary Table 6. Summary results of four SNPs in the GWAS, case-control validation and the combined samples. Supplementary Table 7. Stepwise logistic regression analysis for independent association in GWAS. Supplementary Table 9. Pair-wise LD between HLA-DP alleles tagging SNPs and two representative SNPs (rs and rs ). Supplementary Table 10. Gene expression analysis of the candidate genes between cervical cancer cases and controls from 2 available datasets. Supplementary Table 11. Expression quantitative trait loci (eqtl) analysis of rs , rs and rs for lymphoblastic cell lines of HAPMAP samples. Supplementary Table 12. Association results of 11 positive SNPs in CIN samples. Supplementary Table 14. Oligonucleotides used for HLA-DPA1 and HLA-DPB1 sequencing. 3

4 Supplementary Figure 1. The principal components analysis (PCA) of all initial GWAS samples (1,364 cases and 3,028 controls) and HapMap 270 individuals (JPT+CHB, CEU and YRI) implemented in the software package EIGENSTRAT. Plots of the first two PCs from the PCA of 4,392 samples and 270 HapMap individuals were shown with different colors. 4

5 Supplementary Figure 2. Plots of the first two components derived from a principle component analysis implemented in the software package EIGENSTRAT. (a) the Initial discovery set; (b) the Stringent-matched discovery set. (c) the Matched discovery set; 5

6 Supplementary Figure 3. Quantile-Quantile plots of observed P values in log 10 scale. (a) the Initial discovery set; (b) the Matched discovery set; (c) the Stringent-matched discovery set. 6

7 Supplementary Figure 4. Scatter plot of P values in log 10 scale in the Initial discovery set. 7

8 Supplementary Figure 5. Regional plot of the four identified marker SNPs, including rs at 4q12 (a), rs at 17q12 (b), rs and rs at 6p21.32 (c-d). Results ( log 10 P) are shown for SNPs in the region flanking 150 kb on either side of the marker SNPs (in purple). The genes within the region of interest are shown as arrows. Association results of both genotyped (circles) and imputed (crotch-shaped) SNPs in the Initial discovery set. 8

9 Chr SNP Position Associated Gene Supplementary Table 1. Associations results of 22 SNPs in the first stage of GWAS. a MA Initial discovery study (1,364 cases vs. 3,028 controls) b MAF c P value OR (95% CI) a MA Matched discovery study (1,305cases vs. 1,444 controls) b MAF d P value OR (95% CI) 1p21.1 rs Intergenic C 0.33/ E ( ) C 0.33/ E ( ) 4p15.32 rs PROM1 A 0.15/ E ( ) A 0.15/ E ( ) 4q12 rs EXOC1 T 0.14/ E ( ) T 0.14/ E ( ) 4q12 rs EXOC1 G 0.14/ E ( ) G 0.14/ E ( ) 4q34.3 rs Intergenic C 0.50/ E ( ) C 0.51/ E ( ) 6p21.32 rs HLA-DPA1 C 0.49/ E ( ) T 0.51/ E ( ) 6p21.32 rs HLA-DPB1 G 0.34/ E ( ) G 0.35/ E ( ) 6p21.32 rs HLA-DPB2 A 0.41/ E ( ) A 0.41/ E ( ) 6p21.32 rs HLA-DPB2 A 0.51/ E ( ) A 0.50/ E ( ) 6p21.32 rs HLA-DPB2 G 0.46/ E ( ) A 0.54/ E ( ) 6p21.32 rs HLA-DPB2 C 0.44/ E ( ) C 0.45/ E ( ) 6p21.32 rs COL11A2 A 0.42/ E ( ) A 0.42/ E ( ) 6p21.32 rs Mir C 0.47/ E ( ) T 0.53/ E ( ) 6p21.32 rs Intergenic A 0.39/ E ( ) A 0.40/ E ( ) 8q23.3 rs Intergenic C 0.42/ E ( ) C 0.41/ E ( ) 8q23.3 rs Intergenic G 0.40/ E ( ) G 0.39/ E ( ) 9q22.32 rs FBP2 T 0.20/ E ( ) T 0.20/ E ( ) 10p11.23 rs Intergenic C 0.25/ E ( ) C 0.25/ E ( ) 10q21.3 rs CTNNA3 A 0.33/ E ( ) A 0.33/ E ( ) 14q21.3 rs C14orf28 A 0.13/ E ( ) A 0.13/ E ( ) 17q12 rs Intergenic A 0.29/ E ( ) A 0.29/ E ( ) 17q12 rs GSDMB G 0.29/ E ( ) G 0.29/ E ( ) a MA: Minor allele; b MAF: Minor allele-frequency; c P value: PCA-adjusted P value for the samples in the Initial discovery study; d P value: PCA-adjusted P value for the samples in the Matched discovery study; OR: odds ratio for minor allele; 95% CI: 95% confidence intervals. 9

10 Chr Supplementary Table 2. Associations results of 22 SNPs in the Follow-up 1. SNP Meta-analysis of Follow-up 1 North Center South (1,824 cases vs. 3,808 controls) (600 cases vs. 1,617 controls) (460 cases vs. 968 controls) (764 cases vs. 1,223 controls) a MA P value 1 rs C 2.02E-01 4 rs A 1.72E-01 4 rs T 1.47E-04 4 rs G 2.11E-03 4 rs C 1.99E-01 6 rs T 3.50E-07 6 rs G 4.29E-11 6 rs A 2.03E-04 6 rs A 1.24E-06 6 rs A 4.83E-04 6 rs C 1.11E-08 OR OR OR OR a MA F_A P value a MA F_A P value a MA F_A P value (95% CI) (95% CI) (95% CI) (95% CI) 0.94 ( ) 0.93 ( ) 1.29 ( ) 1.22 ( ) 1.05 ( ) 1.23 ( ) 0.75 ( ) 1.17 ( ) 1.23 ( ) 1.15 ( ) 0.79 ( ) C E-01 A E-01 T E-03 G E-02 C E-01 C E-06 G E-06 A E-02 A E-03 G E-01 C E ( ) 0.91 ( ) 1.36 ( ) 1.25 ( ) 1.04 ( ) 0.73 ( ) 0.71 ( ) 1.17 ( ) 1.25 ( ) 0.93 ( ) 0.82 ( ) C E-01 A E-01 T E-02 G E-01 C E-01 T E-01 G E-05 A E-03 A E-04 A E-06 C E ( ) 0.99 ( ) 1.25 ( ) 1.20 ( ) 1.05 ( ) 1.11 ( ) 0.71 ( ) 1.29 ( ) 1.35 ( ) 1.43 ( ) 0.70 ( ) C E-01 A E-01 T E-02 G E-01 C E-01 T E-03 G E-03 A E-01 A E-02 G E-01 C E ( ) 0.92 ( ) 1.23 ( ) 1.19 ( ) 1.07 ( ) 1.19 ( ) 0.82 ( ) 1.10 ( ) 1.12 ( ) 0.94 ( ) 0.82 ( ) 10

11 6 rs A 1.25E ( ) A E ( ) A E ( ) A E ( ) 6 rs T 3.09E ( ) C E ( ) T E ( ) C E ( ) 6 rs A 4.60E ( ) A E ( ) A E ( ) A E ( ) 8 rs C 2.38E ( ) C E ( ) C E ( ) C E ( ) 8 rs G 9.43E ( ) G E ( ) G E ( ) G E ( ) 9 rs T 6.69E ( ) T E ( ) T E ( ) T E ( ) 10 rs C 7.40E ( ) C E ( ) C E ( ) C E ( ) 10 rs A 9.97E ( ) A E ( ) A E ( ) A E ( ) 14 rs A 2.41E ( ) A E ( ) A E ( ) A E ( ) 17 rs A 8.03E ( ) A E ( ) A E ( ) A E ( ) 17 rs G 4.36E ( ) G E ( ) G E ( ) G E ( ) a MA: Minor allele; OR: odds ratio for minor allele; 95% CI: 95% confidence intervals. 11

12 Supplementary Table 3. Associations results of 13 SNPs in the Follow-up 2. Chr SNP BP A1 F_A F_U A2 P value OR 95% CI 4 rs T C 1.03E rs G A 9.69E rs T C 1.08E rs G T 1.40E rs A G 6.44E rs A G 8.64E rs G A 1.36E rs C T 5.68E rs A C 3.62E rs T C 7.31E rs A G 1.98E rs A G 7.19E rs G A 6.72E BP: Physical position (base-pair); A1: Minor allele; F_A: Frequency of minor allele in cases; F_U: Frequency of minor allele in controls; A2: Major allele; OR: odds ratio for minor allele; 95% CI: 95% confidence intervals. 12

13 Chr Supplementary Table 4. Association results of 13 SNPs in the Matched discovery study, case-control validation and the combined samples. SNP b MA Matched discovery study (1,305cases vs. a Combined_Matched (5,472 cases vs. 1,444 controls) 8,640controls) c P value OR(95% CI) b MA P fix value OR fix (95% CI) P ran value OR ran (95% CI) 4 rs T 4.00E ( ) T 2.82E ( ) 2.82E ( ) 4 rs G 3.71E ( ) G 5.51E ( ) 5.51E ( ) 6 rs T 1.45E ( ) T 2.08E ( ) 1.46E ( ) 6 rs G 3.10E ( ) G 1.26E ( ) 1.26E ( ) 6 rs A 2.45E ( ) A 1.38E ( ) 1.38E ( ) 6 rs A 6.64E ( ) A 1.29E ( ) 1.29E ( ) 6 rs A 6.78E ( ) A 6.96E ( ) 5.51E ( ) 6 rs C 5.14E ( ) C 1.28E ( ) 1.28E ( ) 6 rs A 3.68E ( ) A 4.15E ( ) 1.64E ( ) 6 rs T 3.17E ( ) T 5.86E ( ) 1.28E ( ) 6 rs A 9.70E ( ) A 2.49E ( ) 1.16E ( ) 14 rs A 5.36E ( ) A 6.31E ( ) 2.27E ( ) 17 rs G 5.09E ( ) G 8.51E ( ) 8.51E ( ) a Combined_Matched: Combined results of the Matched discovery study with PCA adjustment and validations; b MA: Minor allele; c P value: PCA-adjusted P value for the samples in the Matched discovery study; P fix value: P value for the fixed effect model; P ran value: P value for the random effect model; OR fix : Odds ratio for the fixed effect model; OR ran : Odds ratio for the random effect model. 13

14 Supplementary Table 5. Association results of 13 SNPs in the Stringent-matched discovery study and the combined samples. Chr SNP Stringent-matched discovery study b MA (829cases vs. 990controls) c P value OR(95% CI) a Combined_Stringent-matched (4,996 cases vs. 8,186controls) b MA P fix value OR fix (95% CI) P ran value OR ran (95% CI) 4 rs T 4.97E ( ) T 9.69E ( ) 9.69E ( ) 4 rs G 5.04E ( ) G 1.41E ( ) 1.41E ( ) 6 rs T 1.50E ( ) T 5.79E ( ) 5.13E ( ) 6 rs G 4.68E ( ) G 4.52E ( ) 1.38E ( ) 6 rs A 4.76E ( ) A 1.32E ( ) 1.32E ( ) 6 rs A 6.15E ( ) A 2.01E ( ) 2.01E ( ) 6 rs G 6.82E ( ) A 2.47E ( ) 1.18E ( ) 6 rs C 4.64E ( ) C 1.64E ( ) 1.64E ( ) 6 rs A 1.86E ( ) A 6.10E ( ) 2.92E ( ) 6 rs T 1.08E ( ) T 1.19E ( ) 3.77E ( ) 6 rs A 8.94E ( ) A 2.31E ( ) 2.88E ( ) 14 rs A 5.09E ( ) A 7.44E ( ) 1.76E ( ) 17 rs G 2.66E ( ) G 2.00E ( ) 2.00E ( ) a Combined_Stringent-matched: Combined results of the Stringent-matched discovery study with PCA adjustment and validations; b MA: Minor allele; c P value: PCA-adjusted P value for the samples in the Stringent-matched discovery study; P fix value: P value for the fixed effect model; P ran value: P value for the random effect model; OR fix : Odds ratio for the fixed effect model; OR ran : Odds ratio for the random effect model. 14

15 Supplementary Table 6. Summary results of four SNPs in the GWAS, case-control validation and the combined samples. SNP Chr Position Locus a MA Stages OR add (95% CI) OR het (95% CI) OR hom (95% CI) rs q EXOC1 T Matched discovery study 1.42 ( ) 1.43 ( ) 1.39 ( ) Stringent-matched dataset 1.34 ( ) 1.34 ( ) 1.36 ( ) b Follow-up ( ) 1.25 ( ) 1.44 ( ) Follow-up ( ) 1.23( ) 1.15( ) c Combined results Matched 1.28 ( ) 1.28 ( ) 1.29 ( ) d Combined results Stringent-matched 1.26 ( ) 1.25 ( ) 1.28 ( ) rs q GSDMB G Matched discovery study 1.29 ( ) 1.25 ( ) 1.31 ( ) Stringent-matched dataset 1.27 ( ) 1.28 ( ) 1.27 ( ) b Follow-up ( ) 1.15 ( ) 1.13 ( ) Follow-up ( ) 1.22( ) 1.17( ) c Combined results Matched 1.19 ( ) 1.20 ( ) 1.18 ( ) d Combined results Stringent-matched 1.18 ( ) 1.20 ( ) 1.16 ( ) rs p HLA-DPB2 G Matched discovery study 0.73 ( ) 0.89 ( ) 0.68 ( ) Stringent-matched dataset 0.66 ( ) 0.83 ( ) 0.62 ( ) b Follow-up ( ) 0.78 ( ) 0.73 ( ) Follow-up ( ) 0.86( ) 0.74( ) c Combined results Matched 0.75 ( ) 0.84 ( ) 0.72 ( ) d Combined results Stringent-matched 0.75 ( ) 0.82 ( ) 0.72 ( ) rs p Intergenic A Matched discovery study 0.78 ( ) 0.74 ( ) 0.78 ( ) Stringent-matched dataset 0.76 ( ) 0.74 ( ) 0.77 ( ) b Follow-up ( ) 0.94 ( ) 0.91 ( ) Follow-up ( ) 0.86( ) 0.82( ) c Combined results Matched 0.85 ( ) 0.86 ( ) 0.84 ( ) d Combined results Stringent-matched 0.85 ( ) 0.87 ( ) 0.84 ( ) a MA: Minor allele; b Follow-up 1: Meta-analysis of Follow-up 1; ORadd: calculated in additive genetic model by logistic regression; ORhet: heterozygote versus common homozygote; ORhom: variant homozygote versus common homozygote; 95% CI: 95% confidence intervals; c Combined results: calculated with the Matched discovery study; d Combined results: calculated with the Stringent-matched discovery study. 15

16 Supplementary Table 7. Stepwise logistic regression analysis for independent association in GWAS. Chr SNP Position Test SNP P value Conditioned on Conditioned SNP P value 1 rs E-05 NA NA 4 rs E rs rs E-07 NA 4 rs E rs E-05 rs NA 4 rs E rs E-05 NA NA 6 rs E rs E rs E rs E rs E rs E rs E-08 NA 6 rs E rs rs E rs E rs E rs E rs E rs E rs E rs E

17 6 rs E rs E rs E rs E-06 NA NA 8 rs E rs E rs E-06 rs NA 8 rs E rs E rs E-06 NA NA 10 rs E-06 NA rs rs E rs E-06 NA NA 14 rs E-05 NA rs rs E rs E rs rs E-06 NA 17 rs E rs rs E-06 NA 17

18 Supplementary Table 9. Pair-wise LD between HLA-DP alleles tagging SNPs and two representative SNPs (rs and rs ). HLA Tag SNP rs rs R2 D' R2 D' P value Haplotype Freq OR Stat P value DPA1*0103 rs E-03 3, E-03 rs E-01 DPA1*0201 rs E-01 1, E-01 rs E-03 DPA1*0202 rs E-05 2, E E-02 DPA1*0401 rs E-01 2, E E-03 DPB1*03:01 rs E-07 1, E-02 rs E-01 DPB1*04:01 rs E-03 4, E-03 rs E-03 DPB1*05:01 rs E-02 3, E-09 rs E-06 DPB1*13:01 rs E-01 4, E-01 rs E-01 DPB1*17:01 rs E-01 3, E-01 rs E-02 DPB1*19:01 rs E-01 4, E-01 rs E-01 OR: odds ratio for minor allele; Stat: Test statistic (T). 18

19 Supplementary Table 10. Gene expression analysis of the candidate genes between cervical cancer cases and controls from 2 available datasets. Pubmed GEO ID Gene Probe_ID FC Regulation P value Sample Description Platform GSE7803 EXOC _s_at 1.02 DOWN normal squamous cervical epitheilia GSDMB _at 1.03 UP HLA-DPB _s_at 1.04 DOWN RING _s_at 1.01 DOWN VPS _s_at 1.01 UP samples and 21 invasive squamous cell carcinomas of the cervix samples GSE9750 EXOC _s_at 1.60 DOWN 1.69E normal squamous cervical epitheilia FC: fold change. GSDMB _at 1.74 UP HLA-DPB _s_at 1.16 DOWN RING _s_at 1.09 DOWN VPS _s_at 1.31 DOWN samples and 33 invasive squamous cell carcinomas of the cervix samples Human Genome U133 Plus 2.0 Array Affymetrix Human Genome U133A Array 19

20 Supplementary Table 11. Expression quantitative trait loci (eqtl) analysis of rs , rs and rs for lymphoblastic cell lines of HAPMAP samples. HAPMAP-FEMALES CHB+JPT-FEMALES CEU-FEMALES YRI-FEMALES SNP Gene Probe BETA P value BETA P value BETA P value BETA P value rs EXOC1 GI_ A -9.77E E E E E E E E-01 CEP135 GI_ S 1.96E E E E E E E E-01 rs GSDMB GI_ S -1.07E E E E E E E E-01 ZPBP2 GI_ I -3.08E E E E E E E E-02 ORMDL3 GI_ S -2.05E E E E E E E E-02 GSDMA GI_ S 1.25E E E E E E E E-01 IKZF3 GI_ A 6.46E E E E E E E E-01 PSMD3 GI_ S 2.84E E E E E E E E-01 rs VPS52 GI_ A -2.02E E E E E E-01 NA NA RING1 GI_ S -3.67E E E E E E-04 NA NA RPS18 GI_ S 2.70E E E E E E-01 NA NA HSD17B8 GI_ S -5.82E E E E E E-02 NA NA B3GALT4 GI_ S -4.98E E E E E E-01 NA NA SLC39A7 GI_ S 5.62E E E E E E-02 NA NA WDR46 GI_ S 3.43E E E E E E-01 NA NA RXRB GI_ S -2.74E E E E E E-01 NA NA COL11A2 GI_ A -3.57E E E E E E-01 NA NA 20

21 Supplementary Table 12. Association results of 11 positive SNPs in CIN samples. a Control vs. CIN a Control vs. CIN + b Cervical Cancer Chr. SNP c MA (7,196 controls vs. 729 cases) (7,196 controls vs. 4,896 cases) d F_A P value OR (95% CI) c MA d F_A P value OR (95% CI) 4 rs T E ( ) T E ( ) 6 rs T E ( ) T E ( ) 6 rs G E ( ) G E ( ) 6 rs A E ( ) A E ( ) 6 rs A E ( ) A E ( ) 6 rs A E ( ) G E ( ) 6 rs C E ( ) C E ( ) 6 rs A E ( ) A E ( ) 6 rs T E ( ) C E ( ) 6 rs A E ( ) A E ( ) 17 rs G E ( ) G E ( ) a Control: control samples from the Follow-up 1 and 2 stages; b Cervical Cancer: cases from the Follow-up 1 and 2 stages; c MA: Minor allele; d F_A: Frequency of minor allele in cases. 21

22 Supplementary Table 14. Oligonucleotides used for HLA-DPA1 and HLA-DPB1 sequencing. Primer 5' sense primer(5'-3') 3' antisense primer(5'-3') HLA-DPA1 (exon 2) ACA TTT TGT CGT GTT TTT CTC T CTC TCA TCC CTT CCA GTT G HLA-DPB1 (exon 2) GAG AGT GGC GCC TCC GCT CAT GCC GGC CCA AAG CCC TCA CTC 22

23 Legends to excel files provided separately Supplementary Table 8. Pair-wise LD between HLA alleles (A, B, C, DQ, DR) tagging SNPs and two representative SNPs (rs and rs ). Supplementary Table 13. GWAS results of SNPs in previously reported candidate genes. 23